Molecules of the HKID-1-related protein family and uses thereof

ABSTRACT

Novel HKID-1 polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length HKID-1 proteins, the invention further provides isolated HKID-1 fusion proteins, antigenic peptides and anti-HKID-1 antibodies. The invention also provides HKID-1 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which an HKID-1 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

BACKGROUND OF THE INVENTION

Protein kinases play critical roles in the regulation of biochemical andmorphological changes associated with cellular growth and division(D'Urso, G. et al. (1990) Science 250: 786-791; Birchmeier. C. et al.(1993) Bioessays 15: 185-189). They serve as growth factor receptors andsignal transducers and have been implicated in cellular transformationand malignancy (Hunter, T. et al. (1992) Cell 70: 375-387; Posada, J. etal. (1992) Mol. Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79:573-582). For example, protein kinases have been shown to participate inthe transmission of signals from growth-factor receptors (Sturgill, T.W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991) Nature353: 170-173), control of entry of cells into mitosis (Nurse, P. (1990)Nature 344: 503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3:269-275) and regulation of actin bundling (Husain-Chishti, A. et al.(1988) Nature 334: 718-721). Protein kinases can be divided into twomain groups based on either amino acid sequence similarity orspecificity for either serine/threonine or tyrosine residues. A smallnumber of dual-specificity kinases are structurally like theserine/threonine-specific group. Within the broad classification,kinases can be further sub-divided into families whose members share ahigher degree of catalytic domain amino acid sequence identity and alsohave similar biochemical properties. Most protein kinase family membersalso share structural features outside the kinase domain that reflecttheir particular cellular roles. These include regulatory domains thatcontrol kinase activity or interaction with other proteins (Hanks, S. K.et al. (1988) Science 241: 42-52).

Rat KID-1 is a serine/threonine protein kinase that is induced bymembrane depolarization or forskolin but not by neurotrophins or growthfactors (Feldman, J. D. et al. (1998). J. Biol. Chem. 273:16535-16543).Rat KID-1 is induced in specific regions of the hippocampus and cortexin response to kainic acid and electroconvulsive shock, suggesting thatrat KID-1 is involved in neuronal function, synaptic plasticity,learning, and memory as well as kainic acid seizures and some nervoussystem-related diseases such as seizures and epilepsy. Rat KID-1paralogs include the PIM-1 proteins known to be proto-oncogenes. Thepresent invention is based, at least in part, on the discovery of thehuman species ortholog of rat KID-1, termed HKID-1.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery of agene encoding HKID-1, an intracellular protein that is predicted to be amember of the serine/threonine protein kinase superfamily. Based onthis, the present invention provides isolated HKID-1 proteins andnucleic acid molecules encoding HKID-1 proteins. The present inventionalso provides methods of detecting HKID-1 protein or HKID-1 nucleicacids and methods for identifying modulators of HKID-1 protein or HKID-1nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sequence (SEQ ID NO:1) and predicted amino acidsequence (SEQ ID NO:2) of human HKID-1. The open reading frame of SEQ IDNO:1 extends from nucleotide 171 to nucleotide 1259 (SEQ ID NO:3).

FIG. 2 depicts an alignment of a portion of the amino acid sequence ofHKID-1 (corresponds to amino acids 40 to 293 of SEQ ID NO:2) and aeukaryotic protein kinase domain consensus sequence derived from ahidden Markov model (PF00069; SEQ ID NO:4). The upper sequence in thealignment is the PF00069 sequence while the lower sequence is amino acid40 to amino acid 293 of SEQ ID NO:2.

FIG. 3 shows a Protean analysis of the HKID-1 amino acid sequence of SEQID NO:2. Shown are: alpha, beta, turn and coil regions identified withthe Garnier-Robson algorithm; alpha, beta and turn regions identifiedwith the Chou-Fasman algorithm; hydrophilicity and hydrophobicity plotsgenerated with the Kyte-Doolittle algorithm; alpha amphipathic and betaamphipathic regions identified with the Eisenberg algorithm; flexibleregions identified with the Karplus-Schulz algorithm; the antigenicindex calculated using the Jameson-Wolf algorithm; and a surfaceprobability plot calculated using the Emini algorithm. For thehydrophobicity plot, relative hydrophobicity is shown above the dottedline, and relative hydrophilicity is shown below the dotted line.

FIG. 4 shows a polypeptide sequence alignment, carried out with theMegAlign program of the DNASTAR sequence analysis package using the J.Hein method with a PAM250 residue weight table, of the HKID-1polypeptide sequence of SEQ ID NO:2 and rat KID-1 (AF086624; SEQ IDNO:5), Xenopus laevis (frog) PIM-1 (Q91822; SEQ ID NO:6), murine PIM-1(P06803; SEQ ID NO:7), rat PIM-1 (P26794; SEQ ID NO:8), and human PIM-1(P11309; SEQ ID NO:9).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of a cDNA moleculeencoding human HKID-1, a member of the serine/threonine kinasesuperfamily. A nucleotide sequence encoding a human HKID-1 protein isshown in FIG. 1 (SEQ ID NO:1; SEQ ID NO:3 includes the open readingframe only). A predicted amino acid sequence of HKID-1 protein is alsoshown in FIG. 1 (SEQ ID NO:2).

The HKID-1 protein of SEQ ID NO:2 is predicted to possess the followingsites or domains: one cAMP- and cGMP-dependent protein kinasephosphorylation site (PS00004) from amino acids 260-263 of SEQ ID NO:2;three protein kinase C phosphorylation sites (PS00005) from amino acids137-139, 275-277, and 279-281, of SEQ ID NO:2; three casein kinase IIphosphorylation sites (PS00006) from amino acids 202-205, 211-214, and321-324, of SEQ ID NO:2; one tyrosine kinase phosphorylation site(PS00007) from amino acid 33-40, of SEQ ID NO:2; SEQ ID NO:15; sevenN-myristoylation sites (PS00008) from amino acids 43-48, 49-54, 57-62,63-68, 80-85, 98-103, and 295-300 of SEQ ID NO:2; one protein kinaseATP-binding region signature (PS00107) from amino acid 46-54, of SEQ IDNO:2; one serine/threonine protein kinase active site signature(PS00108) from amino acid 166-178, of SEQ ID NO:2; and one eukaryoticprotein kinase domain consensus derived from a hidden Markov model (HMM)(PF00069; SEQ ID NO:4) from amino acid 40-293, of SEQ ID NO:2. Forgeneral information regarding PFAM identifiers, PS prefix and PF prefixdomain identification numbers, refer to Sonnhammer et al. (1997) Protein28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html.

The HKID-1 polypeptide sequence of SEQ ID NO:2 was analyzed with theMEMSAT transmembrane domain prediction software. MEMSAT predicted threepotential transmembrane domains in the HKID-1 polypeptide sequence ofSEQ ID NO:2: amino acid 42 to 58, amino acid 78 to 94, and amino acid226 to 245. Because the rat ortholog of HKID-1, rat KID-1, is known tobe a soluble protein, it is likely that the potential transmembranedomains predicted by MEMSAT represent hydrophobic domains of HKID-1protein involved in hydrophobic interactions in the core of the HKID-1protein and not transmembrane domains.

In an embodiment of the invention, the HKID-1 molecules are proteinkinases which are expressed and/or function in cells of the nervoussystem, as a nonlimiting example, cells of the hippocampus and cortex.

As used herein, the term "protein kinase" includes a protein orpolypeptide which is capable of modulating its own phosphorylation stateor the phosphorylation state of another protein or polypeptide. Proteinkinases can have a specificity for (i.e., a specificity tophosphorylate) serine/threonine residues, tyrosine residues, or bothserine/threonine and tyrosine residues, e.g., the dual specificitykinases. Specificity of a protein kinase for phosphorylation of eithertyrosine or serine/threonine can be predicted by the sequence of two ofthe subdomains, VIb and VIII, (described in, for example, Hanks et al.(1988) Science 241:42-52, the contents of which are incorporated hereinby reference).

Protein kinases play a role in signalling pathways associated with cellsexpressing them. Thus, since the HKID-1 molecules are expressed inneuronal cells, HKID-1 may be involved in: 1) nervous system disorders;2) seizures; 3) epilepsy; 4) learning; 5) memory; or 6) synapticplasticity. HKID-1 may also be involved in proliferative disorders, suchas cancer, because HKID-1 is the paralog of the PIM-1 proteins which areknown to be proto-oncogenes.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

The HKID-1 cDNA sequence (SEQ ID NO:1), which is approximately 2126nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of 978 base pairs (nucleotides171-1259 of SEQ ID NO:1; SEQ ID NO:3) encoding a 326 amino acid protein(SEQ ID NO:2) having a predicted molecular weight of approximately 35.86kDa (excluding post-translational modifications) (FIG. 1).

One aspect of the invention provides isolated nucleic acid moleculesthat encode HKID-1 proteins or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify HKID-1-encoding nucleic acids (e.g., HKID-1 mRNA) andfragments for use as PCR primers for the amplification or mutation ofHKID-1 nucleic acid molecules. As used herein, the term "nucleic acidmolecule" is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An "isolated" nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an "isolated" nucleic acid is free ofsequences (preferably protein encoding sequences) which naturally flankthe nucleic acid (i.e., sequences located at the 5' and 3' ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatedHKID-1 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. Moreover, an "isolated" nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

An isolated nucleic acid molecule of the present invention, e.g., anucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, SEQID NO:3, or a complement of any of these nucleotide sequences, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. Using all or a portion of the nucleic acidsequences of SEQ ID NO:1 or SEQ ID NO:3, HKID-1 nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook et al., eds., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to HKID-1 nucleotide sequences can be prepared by standardsynthetic techniques, using an automated DNA synthesizer.

The invention features an isolated nucleic acid molecule which is atleast 26% (or 30%, 35%, 40%, 45%, 55%, 65%, 75%, 85%, 95%, or 98%)identical to the nucleotide sequence shown in SEQ ID NO: 1 or acomplement thereof. The invention also features an isolated nucleic acidmolecule which is at least 43% (or 45%, 50%, 55%, 65%, 75%, 85%, 95%, or98%) identical to the nucleotide sequence shown in SEQ ID NO:3 or acomplement thereof.

The invention also features an isolated nucleic acid molecule whichincludes a nucleotide sequence encoding a protein having an amino acidsequence that is at least 95.5% (or 95.8%, 96%, 96.5%, 97%, 98% or 99%)identical to the amino acid sequence of SEQ ID NO:2.

In an embodiment, an isolated HKID-1 nucleic acid molecule has thenucleotide sequence shown SEQ ID NO:1 or SEQ ID NO:3.

Also within the invention is an isolated nucleic acid molecule whichencodes a fragment of a polypeptide having the amino acid sequence ofSEQ ID NO:2, the fragment including at least 15 (or 25, 30, 50, 100,150, 200, 250, 270, 290, 310 or 326) contiguous amino acids of SEQ IDNO:2.

Moreover, the isolated nucleic acid molecule of the invention cancomprise only a portion of an isolated nucleic acid sequence encodingHKID-1, for example, a fragment which can be used as a probe or primeror a fragment encoding a biologically active portion of HKID-1, forexample, fragments comprising nucleotides 306 to 332 of SEQ ID SEQ IDNO:1, encoding the protein kinase ATP-binding region signature domain ofHKID-1, nucleotides 666 to 704 of SEQ ID NO:1, encoding theserine/threonine protein kinase active site signature domain of HKID-1,and nucleotides 288 to 1049 of SEQ ID NO:1 encoding the eukaryoticprotein kinase domain of HKID-1.

The nucleotide sequence determined from the human HKID-1 gene and/orcDNA allows for the generation of probes and primers designed for use inidentifying and/or cloning HKID-1 homologs in other cell types, e.g.,from other tissues, as well as HKID-1 orthologs and homologs from othermammals. The probe/primer typically comprises a substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of thesense or anti-sense sequence of SEQ ID NO:1 or SEQ ID NO:3 or of anaturally occurring mutant and/or allelelic variant of SEQ ID NO:1 orSEQ ID NO:3.

Probes based on the human HKID-1 nucleotide sequence can be used todetect transcripts, cDNAs, or genomic sequences encoding the same oridentical proteins or allelic variants thereof. The probe comprises alabel group attached thereto, e.g., a radioisotope, a fluorescentcompound, an enzyme, or an enzyme co-factor. Such probes can be used aspart of a diagnostic test kit for identifying cells or tissues whichmis-express an HKID-1 protein, such as by measuring levels of anHKID-1-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting HKID-1 mRNA levels or determining whether a genomic HKID-1gene has been mutated or deleted.

Another embodiment of the invention features isolated HKID-1 nucleicacid molecules which specifically detect HKID-1 nucleic acid moleculesrelative to nucleic acid molecules encoding other members of theserine/threonine protein kinase superfamily. For example, in oneembodiment, an isolated HKID-1 nucleic acid molecule hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complementthereof. In another embodiment, an isolated HKID-1 nucleic acid moleculeis at least 547 (or 550, 600, 650, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2126 or 2200)nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3, or a complement thereof. In another embodiment, anisolated HKID-1 nucleic acid molecule comprises nucleotides 306 to 332of SEQ ID NO:1, encoding the protein kinase ATP-binding region signaturedomain of HKID-1, or a complement thereof. In yet another embodiment, anisolated HKID-1 nucleic acid molecule comprises nucleotides 666 to 704of SEQ ID NO:1, encoding the serine/threonine protein kinase active sitesignature domain of HKID-1, or a complement thereof. In anotherembodiment, an isolated HKID-1 nucleic acid molecule comprisesnucleotides 288 to 1049 of SEQ ID NO:1 encoding the eukaryotic proteinkinase domain of HKID-1, or a complement thereof. In another embodiment,the invention provides an isolated nucleic acid molecule which isantisense to the coding strand of an HKID-1 nucleic acid.

An isolated nucleic acid fragment encoding a "biologically activeportion of HKID-1" can be prepared by isolating a portion of SEQ ID NO:1or SEQ ID NO:3, expressing the encoded portion of HKID-1 protein (e.g.,by recombinant expression in vitro) and assessing the activity of theencoded portion of HKID-1. For example, an isolated nucleic acidfragment encoding a biologically active portion of HKID-1 includes oneor more of a cAMP- and cGMP-dependent protein kinase phosphorylationsite (PS00004), for example, from amino acids 260-263 of SEQ ID NO:2; aprotein kinase C phosphorylation site (PS00005), for example, from aminoacids 137-139, 275-277, and 279-281, of SEQ ID NO:2; a casein kinase IIphosphorylation site (PS00006), for example, from amino acids 202-205,211-214, and 321-324, of SEQ ID NO:2; a tyrosine kinase phosphorylationsite (PS00007), for example, from amino acid 33-40, of SEQ ID NO:2; anN-myristoylation sites (PS00008) from amino acids 43-48, 49-54, 57-62,63-68, 80-85, 98-103, and 295-300 of SEQ ID NO:2; a protein kinaseATP-binding region signature (PS00107), for example, from amino acid46-54, of SEQ ID NO:2; a serine/threonine protein kinase active sitesignature (PS00108), for example, from amino acid 166-178, of SEQ IDNO:2; and a eukaryotic protein kinase domain (PF00069; SEQ ID NO:4), forexample, from amino acid 40-293, of SEQ ID NO:2.

The invention further encompasses isolated nucleic acid molecules thatdiffer from the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, dueto degeneracy of the genetic code and thus encode the same HKID-1protein as that encoded by the nucleotide sequence shown in SEQ ID NO:1or SEQ ID NO:3.

In addition to the human HKID-1 nucleotide sequence shown in SEQ ID NO:1or SEQ ID NO:3, it will be appreciated by those skilled in the art thatDNA sequence polymorphisms that lead to changes in the amino acidsequences of HKID-1 may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the HKID-1 gene may existamong individuals within a population due to natural allelic variation.An allele is one of a group of genes which occur alternatively at agiven genetic locus. As used herein, the terms "gene" and "recombinantgene" refer to nucleic acid molecules comprising an open reading frameencoding an HKID-1 protein, preferably a mammalian HKID-1 protein. Asused herein, the phrase "allelic variant" refers to a nucleotidesequence which occurs at an HKID-1 locus or to a polypeptide encoded bythe nucleotide sequence. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of the HKID-1 gene.Alternative alleles can be identified by sequencing the gene of interestin a number of different individuals. This can be readily carried out byusing hybridization probes to identify the same genetic locus in avariety of individuals. Any and all such nucleotide variations andresulting amino acid polymorphisms or variations in HKID-1 that are theresult of natural allelic variation and that do not alter the functionalactivity of HKID-1 are intended to be within the scope of the invention.Allelic variants of HKID-1 will physically and genetically map to theHKID-1 genetic and physical locus shown in Example 5 to be chromosome 22between the D22S1169 and D22S₋₋ qter markers, 196.70 centirays from thetop of the chromosome 22 linkage group.

The invention includes an isolated nucleic acid molecule which encodes anaturally occurring allelic variant, encoding a fully functionalprotein, a partially functional HKID-1 protein, or a non functionalprotein, of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, wherein the nucleic acid molecule hybridizes to a nucleic acidmolecule comprising SEQ ID NO:1, SEQ ID NO:3 or a complement thereofunder stringent conditions.

Moreover, isolated nucleic acid molecules encoding HKID-1 proteins fromother species (HKID-1 homologs or orthologs), which have a nucleotidesequence which differs from that of a human HKID-1, are intended to bewithin the scope of the invention, excluding those known in the art,e.g., the rat and Xenopus laevis (frog) species orthologs of HKID-1.Nucleic acid molecules corresponding to natural allelic variants,homologs, and orthologs of the HKID-1 cDNA of the invention can beisolated based on their identity to the human HKID-1 nucleic acidsdisclosed herein using the human cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. Orthologs of HKID-1 will often mapto genetic loci that are syntenic with the human HKID-1 genetic andphysical locus shown in Example 5 to be chromosome 22 between theD22S1169 and D22S₋₋ qter markers, 196.70 centiRays from the top of thechromosome 22 linkage group.

In another embodiment of the invention, an isolated nucleic acidmolecule of the invention is 1) at least 547 (or 550, 600, 650, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100 or 2126) nucleotides of the nucleotide sequence shown in SEQID NO:1; or 2) at least 415 (or 450, 500, 550, 600, 650, 700, 800, 900or 978) nucleotides of the nucleotide sequence shown in SEQ ID NO:3; or3) at least 8 (or 10, 15, 20, 25, 35, 45, 65, 85, 105, 125, 175, 225,275, 325, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or923) nucleotides from nucleotide 1-923 of SEQ ID NO:1) at least 8 (or10, 15, 20, 25, 35, 45, 65, 85, 105, 125, 175, 225, 275, 325 or 344)nucleotides from nucleotide 1-344 of SEQ ID NO:3 and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence, preferably the coding sequence, of SEQ ID SEQ IDNO:3, or a complement thereof.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or a portionthereof. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence under stringent conditions.

As used herein, the term "hybridizes under stringent conditions" isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A non-limiting example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50-65° C. Preferably, an isolated nucleic acid molecule of the inventionthat hybridizes under stringent conditions to the sequence of SEQ IDNO:1, SEQ ID NO:3, or the complement thereof, corresponds to anaturally-occurring nucleic acid molecule. As used herein, a"naturally-occurring" nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

In addition to naturally-occurring allelic variants of the HKID-1sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, thereby leading tochanges in the amino acid sequence of the encoded HKID-1 protein,without altering the biological activity of the HKID-1 protein. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at "non-essential" amino acid residues. A "non-essential"amino acid residue is a residue that can be altered from the wild-typesequence of HKID-1 (e.g., the sequence of SEQ ID NO:2) without alteringthe biological activity, whereas an "essential" amino acid residue isrequired for biological activity. For example, amino acid residues thatare not conserved or only semi-conserved among HKID-1 of various speciesmay be non-essential for activity and thus would be likely targets foralteration. Alternatively, amino acid residues that are conserved amongthe HKID-1 proteins of various species may be essential for activity andthus would not be likely targets for alteration.

For example, HKID-1 proteins of the present invention contain at leastone conserved protein kinase ATP-binding region signature (PS00107) fromamino acid 46-54, of SEQ ID NO:2; at least one conservedserine/threonine protein kinase active site signature (PS00108) fromamino acid 166-178, of SEQ ID NO:2; and at least one conservedeukaryotic protein kinase domain (PF00069) from amino acid 40-293, ofSEQ ID NO:2. For example, HKID-1 proteins of the present invention maycontain at least one conserved or nonconserved cAMP- and cGMP-dependentprotein kinase phosphorylation site (PS00004), for example, from aminoacids 260-263 of SEQ ID NO:2; protein kinase C phosphorylation site(PS00005), for example, from amino acids 137-139, 275-277, and 279-281,of SEQ ID NO:2; casein kinase II phosphorylation site (PS00006), forexample, from amino acids 202-205, 211-214, and 321-324, of SEQ ID NO:2;tyrosine kinase phosphorylation site (PS00007), for example, from aminoacid 33-40, of SEQ ID NO:2; N-myristoylation site (PS00008), forexample, from amino acids 43-48, 49-54, 57-62, 63-68, 80-85, 98-103, and295-300 of SEQ ID NO:2.

Accordingly, another aspect of the invention provides nucleic acidmolecules encoding HKID-1 proteins that contain changes in amino acidresidues that are not essential for activity. Such HKID-1 proteinsdiffer in amino acid sequence from SEQ ID NO:2 yet retain biologicalactivity. In one embodiment, the isolated nucleic acid molecule includesa nucleotide sequence encoding a protein that includes an amino acidsequence that is at least about 45%, 55%, 65%, 75%, 85%, 95%, or 98%identical to the amino acid sequence of SEQ ID NO:2.

An isolated nucleic acid molecule encoding an HKID-1 protein having asequence which differs from that of SEQ ID NO:2 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A"conservative amino acid substitution" is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in HKID-1 is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, mutations can be introduced randomly along all orpart of an HKID-1 coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for HKID-1 biological activityto identify mutants that retain activity. Following mutagenesis, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

In an embodiment, a mutant HKID-1 can be assayed for (1) the ability tobe phosphorylated by protein kinases, (2) the ability to beN-myristoylated, (3) the ability to bind ATP, (4) the ability tophosphorylate proteins, and (5) the ability to phosphorylate proteinsspecifically on serine and threonine residues. In another embodiment,mutant HKID-1 can be assayed for its ability to play a role insignalling pathways associated with cells that express HKID-1, e.g.cells of the nervous system, the ability to form protein-proteininteraction with its substrate proteins expressed in cells in whichHKID-1 is expressed, and the ability to form protein-proteininteractions with proteins in the signal transduction and biologicalpathways that exist in cells in which HKID-1 is expressed.

The present invention further encompasses antisense nucleic acidmolecules, i.e., molecules which are complementary to a sense nucleicacid encoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire HKID-1 coding strand, or to only a portion thereof, e.g., all orpart of the protein coding region (or open reading frame). An antisensenucleic acid molecule can be antisense to a noncoding region of thecoding strand of a nucleotide sequence encoding HKID-1. The noncodingregions ("5' and 3' untranslated regions") are the 5' and 3' sequenceswhich flank the coding region and are not translated into amino acids.

Given the coding strand sequences encoding HKID-1 disclosed herein(e.g., SEQ ID NO:1 or SEQ ID NO:3), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of HKID-1 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of HKID-1 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of HKID-1 mRNA, e.g., an oligonucleotide havingthe sequence AGAGCAGCATCGCGGGCGACGGC (SEQ ID NO:10) orAGCAGCATCGCGGGCGAC (SEQ ID NO:11). An antisense oligonucleotide can be,for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotidesin length. An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5'-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an HKID-1protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2'-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave HKID-1 mRNA transcripts to thereby inhibittranslation of HKID-1 mRNA. A ribozyme having specificity for anHKID-1-encoding nucleic acid can be designed based upon the nucleotidesequence of an HKID-1 cDNA disclosed herein (e.g., SEQ ID NO:1, SEQ IDNO:3). For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in anHKID-1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, HKID-1 mRNA canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel and Szostak(1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, HKID-1 gene expression can be inhibitedby targeting nucleotide sequences complementary to the regulatory regionof the HKID-1 (e.g., the HKID-1 promoter and/or enhancers) to formtriple helical structures that prevent transcription of the HKID-1 genein target cells. See generally Helene (1991) Anticancer Drug Des.6(6):569; Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992)Bioassays 14(12):807.

In embodiments, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Nati. Acad. Sci.USA 93:14670.

PNAs of HKID-1 can be used in therapeutic and diagnostic applications.For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofHKID-1 can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup (1996), supra; or as probes or primers for DNAsequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al.(1996), supra).

In another embodiment, PNAs of HKID-1 can be modified, e.g., to enhancetheir stability, specificity or cellular uptake, by attaching lipophilicor other helper groups to PNA, by the formation of PNA-DNA chimeras, orby the use of liposomes or other techniques of drug delivery known inthe art. The synthesis of PNA-DNA chimeras can be performed as describedin Hyrup (1996), supra, Finn et al. (1996) Nucleic Acids Res.24(17):3357-63, Mag et al. (1989) Nucleic Acids Res. 17:5973, andPeterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

II. Isolated HKID-1 Proteins

One aspect of the invention provides isolated HKID-1 proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-HKID-1 antibodies. In oneembodiment, native HKID-1 proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, HKID-1 proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, an HKID-1 protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theHKID-1 protein is derived, or substantially free of chemical precursorsor other chemicals when chemically synthesized. The language"substantially free of cellular material" includes preparations ofHKID-1 protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, HKID-1 protein that is substantially free of cellularmaterial includes preparations of HKID-1 protein having less than about30%, 20%, 10%, or 5% (by dry weight) of non-HKID-1 protein (alsoreferred to herein as a "contaminating protein"). When the HKID-1protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When HKID-1 protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of HKID-1 protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors ornon-HKID-1 chemicals.

In one embodiment, the isolated proteins of the present invention,preferably HKID-1 proteins, are identified based on the presence in themof at least one "protein kinase ATP-binding site" and at least one"serine/threonine protein kinase active site" and that they have anamino acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 81%, 85%,90%, 95%, 99% or more homologous to an amino acid sequence including SEQID NO:2. As used herein, the term "protein kinase ATP-binding site"includes an amino acid sequence with significant amino acid sequencesimilarity to the protein kinase ATP-binding region signature sequence(PS00107) which is conserved in protein kinases. As used herein, theterm "serine/threonine protein kinase active site" includes an aminoacid sequence with significant amino acid sequence similarity to theserine/threonine protein kinase active site signature sequence (PS00108)which is conserved in protein kinases that phosphorylate serine andthreonine residues on proteins.

In another embodiment, the isolated proteins of the present invention,preferably HKID-1 proteins, are identified based on the presence of atleast one eukaryotic protein kinase domain and that they have an aminoacid sequence which is at least 60%, 65%, 70%, 75%, 80%, 81%, 85%, 90%,95%, 99% or more homologous to an amino acid sequence including SEQ IDNO:2. As used herein, the term "eukaryotic protein kinase domain"includes an amino acid sequence with significant amino acid sequencesimilarity to the eukaryotic protein kinase domain sequence (PF00069) ofSEQ ID NO:4 which is conserved in protein kinases.

Yet another embodiment of the invention includes an isolated HKID-1protein which is encoded by a nucleic acid molecule having a nucleotidesequence that is at least about 43% (or 45%, 50%, 55%, 65%, 75%, 85%,95%, or 98%) identical to SEQ ID NO:3; an isolated HKID-1 protein whichis encoded by a nucleic acid molecule having a nucleotide sequence atleast about 65%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%identical to the portions of SEQ ID NO:1 encoding the cAMP- andcGMP-dependent protein kinase phosphorylation site (PS00004) from aminoacids 260-263 of SEQ ID NO:2; the three protein kinase C phosphorylationsites (PS00005) from amino acids 137-139, 275-277, and 279-281, of SEQID NO:2; the three casein kinase II phosphorylation sites (PS00006) fromamino acids 202-205, 211-214, and 321-324, of SEQ ID NO:2; the tyrosinekinase phosphorylation site (PS00007) from amino acid 33-40, of SEQ IDNO:2; the seven N-myristoylation sites (PS00008) from amino acids 43-48,49-54, 57-62, 63-68, 80-85, 98-103, and 295-300 of SEQ ID NO:2; and anisolated HKID-1 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 65%, preferably 65%, 70%,75%, 80%, 85%, 90%, 95% or 99% identical to the portions of SEQ ID NO:1encoding the protein kinase ATP-binding region signature (PS00107) fromamino acid 46-54, of SEQ ID NO:2; (e.g., about nucleotides 306 to 332 ofSEQ ID NO:1); the serine/threonine protein kinase active site signature(PS00108) from amino acid 166-178, of SEQ ID NO:2 (e.g., aboutnucleotides 666 to 704 of SEQ ID NO:1); and the eukaryotic proteinkinase domain (PF00069; SEQ ID NO:4) from amino acid 40-293, of SEQ IDNO:2 (e.g., about nucleotides 288 to 1049 of SEQ ID NO:1) and anisolated HKID-1 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:3, or the complement thereof.

Biologically active portions of an HKID-1 protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the HKID-1 protein (e.g., the amino acidsequence shown in SEQ ID NO:2), which include fewer amino acids than thefull length HKID-1 proteins, and exhibit at least one activity of anHKID-1 protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the HKID-1 protein. Abiologically active portion of an HKID-1 protein can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.Biologically active polypeptides include one or more identified HKID-1structural domains, e.g., a cAMP- and cGMP-dependent protein kinasephosphorylation site (PS00004), for example, from amino acids 260-263 ofSEQ ID NO:2; a protein kinase C phosphorylation site (PS00005), forexample, from amino acids 137-139, 275-277, and 279-281, of SEQ ID NO:2;a casein kinase II phosphorylation site (PS00006), for example, fromamino acids 202-205, 211-214, and 321-324, of SEQ ID NO:2; a tyrosinekinase phosphorylation site (PS00007), for example, from amino acid33-40, of SEQ ID NO:2; an N-myristoylation site (PS00008), for example,from amino acids 43-48, 49-54, 57-62, 63-68, 80-85, 98-103, and 295-300of SEQ ID NO:2; a protein kinase ATP-binding region signature (PS00107),for example, from amino acid 46-54, of SEQ ID NO:2; a serine/threonineprotein kinase active site signature (PS00108), for example, from aminoacid 166-178, of SEQ ID NO:2; and an eukaryotic protein kinase domain(PF00069), for example, from amino acid 40-293, of SEQ ID NO:2.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeHKID-1 protein.

HKID-1 protein has the amino acid sequence shown of SEQ ID NO:2. Otheruseful HKID-1 proteins are substantially identical to SEQ ID NO:2 andretain the functional activity of the protein of SEQ ID NO:2 yet differin amino acid sequence due to natural allelic variation or mutagenesis.For example, such HKID-1 proteins and polypeptides posses at least onebiological activity described herein such as, (1) the ability to bephosphorylated by protein kinases, (2) the ability to beN-myristoylated, (3) the ability to bind ATP, (4) the ability tophosphorylate proteins, (5) the ability to phosphorylate proteinsspecifically on serine and threonine residues, (6) the ability to play arole in signalling pathways associated with cells that express HKID-1,e.g. cells of the nervous system, (7) the ability to formprotein-protein interaction with its substrate proteins expressed incells in which HKID-1 is expressed, and (8) the ability to formprotein-protein interactions with proteins in the signal transductionand biological pathways that exist in cells in which HKID-1 isexpressed. Accordingly, a useful isolated HKID-1 protein is a proteinwhich includes an amino acid sequence at least about 45%, preferably55%, 65%, 75%, 85%, 95%, or 99% identical to the amino acid sequence ofSEQ ID NO:2 and retains the functional activity of the HKID-1 proteinsof SEQ ID NO:2. In other instances, the HKID-1 protein is a proteinhaving an amino acid sequence 55%, 65%, 75%, 85%, 95%, or 98% identicalto one or more of the HKID-1 domains including one cAMP- andcGMP-dependent protein kinase phosphorylation site (PS00004) from aminoacids 260-263 of SEQ ID NO:2; three protein kinase C phosphorylationsites (PS00005) from amino acids 137-139, 275-277, and 279-281, of SEQID NO:2; three casein kinase II phosphorylation sites (PS00006), fromamino acids 202-205, 211-214, and 321-324, of SEQ ID NO:2; one tyrosinekinase phosphorylation site (PS00007) from amino acid 33-40, of SEQ IDNO:2; seven N-myristoylation sites (PS00008) from amino acids 43-48,49-54, 57-62, 63-68, 80-85, 98-103, and 295-300 of SEQ ID NO:2; oneprotein kinase ATP-binding region signature (PS00107) from amino acid46-54, of SEQ ID NO:2; one serine/threonine protein kinase active sitesignature (PS00108) from amino acid 166-178, of SEQ ID NO:2; and oneeukaryotic protein kinase domain (PF00069; SEQ ID NO:4) from amino acid40-293, of SEQ ID NO:2. In an embodiment, the HKID-1 protein retains afunctional activity of the HKID-1 protein of SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., percentidentity=number of identical positions/total number of positions (e.g.,overlapping positions)×100). In one embodiment, the two sequences arethe same length.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to HKID-1 nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to HKID-1 proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search which detects distant relationshipsbetween molecules. Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides HKID-1 chimeric or fusion proteins. As usedherein, an HKID-1 "chimeric protein" or "fusion protein" comprises anHKID-1 polypeptide operably linked to a non-HKID-1 polypeptide. A"HKID-1 polypeptide" refers to a polypeptide having an amino acidsequence corresponding to HKID-1, whereas a "non-HKID-1 polypeptide"refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially identical to the HKID-1 protein,e.g., a protein which is different from the HKID-1 protein and which isderived from the same or a different organism. Within an HKID-1 fusionprotein the HKID-1 polypeptide can correspond to all or a portion of anHKID-1 protein, preferably at least one biologically active portion ofan HKID-1 protein. Within the fusion protein, the term "operably linked"is intended to indicate that the HKID-1 polypeptide and the non-HKID-1polypeptide are fused in-frame to each other. The non-HKID-1 polypeptidecan be fused to the N-terminus or C-terminus of the HKID-1 polypeptide.

One useful isolated fusion protein is a GST-HKID-1 fusion protein inwhich the HKID-1 sequences are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant HKID-1.

In another embodiment, the fusion protein is an HKID-1 proteincontaining an heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofHKID-1 can be increased through use of a heterologous signal sequence.For example, the gp67 secretory sequence of the baculovirus envelopeprotein can be used as a heterologous signal sequence (Current Protocolsin Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992).Other examples of eukaryotic heterologous signal sequences include thesecretory sequences of melittin and human placental alkaline phosphatase(Stratagene; La Jolla, Calif.). In yet another example, usefulprokaryotic heterologous signal sequences include the phoA secretorysignal (Sambrook et al., supra) and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is anHKID-1-immunoglobulin fusion protein in which all or part of HKID-1 isfused to sequences derived from a member of the immunoglobulin proteinfamily.

Preferably, an HKID-1 chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, e.g., Ausubel et al., supra). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). An HKID-1-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the HKID-1 protein.

The present invention also provides variants of the HKID-1 proteins(i.e., proteins having a sequence which differs from that of the HKID-1amino acid sequence). Such variants can function as either HKID-1agonists (mimetics) or as HKID-1 antagonists. Variants of the HKID-1protein can be generated by mutagenesis, e.g., discrete point mutationor truncation of the HKID-1 protein. An agonist of the HKID-1 proteincan retain substantially the same, or a subset, of the biologicalactivities of the naturally occurring form of the HKID-1 protein, e.g.,(1) the ability to be phosphorylated by protein kinases, (2) the abilityto be N-myristoylated, (3) the ability to bind ATP, (4) the ability tophosphorylate proteins, (5) the ability to phosphorylate proteinsspecifically on serine and threonine residues, (6) the ability to play arole in signalling pathways associated with cells that express HKID-1,e.g. cells of the nervous system, (7) the ability to formprotein-protein interaction with its substrate proteins expressed incells in which HKID-1 is expressed, and (8) the ability to formprotein-protein interactions with proteins in the signal transductionand biological pathways that exist in cells in which HKID-1 isexpressed. An antagonist of the HKID-1 protein can inhibit one or moreof the activities of the naturally occurring form of the HKID-1 proteinby, for example, competitively binding to a downstream or upstreammember of a cellular signaling cascade which includes the HKID-1protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein can have fewer side effects in a subjectrelative to treatment with the naturally occurring form of the HKID-1proteins.

Variants of the HKID-1 protein which function as either HKID-1 agonists(mimetics) or as HKID-1 antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theHKID-1 protein for HKID-1 protein agonist or antagonist activity. In oneembodiment, a variegated library of HKID-1 variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of HKID-1 variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential HKID-1 sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of HKID-1 sequences therein. There are avariety of methods which can be used to produce libraries of potentialHKID-1 variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential HKID-1 sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

In addition, libraries of fragments of the HKID-1 protein codingsequence can be used to generate a variegated population of HKID-1fragments for screening and subsequent selection of variants of anHKID-1 protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofan HKID-1 coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal and internal fragments of various sizes of the HKID-1protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of HKID-1 proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify HKID-1variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Also within the invention is an isolated polypeptide which is anaturally occurring allelic variant, comprising a fully functionalprotein, a partially functional protein, or a non functional protein, ofa polypeptide that includes the amino acid sequence of SEQ ID NO:2,wherein the polypeptide is encoded by a nucleic acid molecule whichhybridizes to a nucleic acid molecule comprising SEQ ID NO:1, SEQ IDNO:3 or a complement thereof under stringent conditions. The allelicvariants of HKID-1 will be encoded by a gene that will physically andgenetically map to the HKID-1 genetic and physical locus shown inExample 5 to be chromosome 22 between the D22S1169 and D22S₋₋ qtermarkers, 196.70 centiRays from the top of the chromosome 22 linkagegroup.

Also within the invention is an isolated polypeptide which is a speciesortholog of HKID-1, a polypeptide that includes the amino acid sequenceof SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acidmolecule which hybridizes to a nucleic acid molecule comprising SEQ IDNO:1, SEQ ID NO:3 or a complement thereof under stringent conditions.Species orthologs of HKID-1 will often physically and genetically map tothe region of the genome of the species from which they originate thatis syntenic to human chromosome 22 between the D22S1169 and D22S₋₋ qtermarkers, 196.70 centiRays from the top of the chromosome 22 linkagegroup.

III. Anti-HKID-1 Antibodies

The present invention further provides antibodies that bind to theHKID-1 proteins of the present invention. The term "antibody" as usedherein refers to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site which specifically binds an antigen, such asHKID-1. A molecule which specifically binds to HKID-1 is a moleculewhich binds HKID-1, but does not substantially bind other molecules in asample, e.g., a biological sample, which naturally contains HKID-1.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab')₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind HKID-1. The term"monoclonal antibody" or "monoclonal antibody composition", as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of HKID-1. A monoclonal antibody composition thustypically displays a single binding affinity for a particular HKID-1protein with which it immunoreacts.

An isolated HKID-1 protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind HKID-1 usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length HKID-1 protein can be used or, alternatively, theinvention provides antigenic peptide fragments of HKID-1 for use asimmunogens. The antigenic peptide of HKID-1 comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence shown in SEQ ID NO:2 and encompasses an epitope of HKID-1 suchthat an antibody raised against the peptide forms a specific immunecomplex with HKID-1.

Epitopes encompassed by the antigenic peptide are regions of HKID-1 thatare located on the surface of the protein. A surface probabilityanalysis, presented in FIG. 3, of the polypeptide sequence (SEQ ID NO:2)of human HKID-1 protein identifies probable antigenic regions; aminoacid 28 to 39, amino acid 124 to 129, and amino acid 277 to 283 areparticularly likely to be localized to the surface of the protein and,therefore, are likely to encode surface residues useful for targetingantibody production.

AN HKID-1 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed HKID-1 protein or achemically synthesized HKID-1 polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic HKID-1 preparation induces a polyclonal anti-HKID-1antibody response.

Polyclonal anti-HKID-1 antibodies can be prepared as described above byimmunizing a suitable subject with an HKID-1 immunogen. The anti-HKID-1antibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized HKID-1. If desired, the antibody moleculesdirected against HKID-1 can be isolated from the mammal (e.g., from theblood) and further purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-HKID-1 antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing hybridomasis well known (see generally Current Protocols in Immunology (1994)Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly,an immortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with an HKID-1 immunogenas described above, and the culture supernatants of the resultinghybridoma cells are screened to identify a hybridoma producing amonoclonal antibody that binds HKID-1.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-HKID-1 monoclonal antibody (see, e.g., Current Protocols inImmunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth,in Monoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol.Med., 54:387-402. Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line, e.g., a myeloma cell line that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine ("HAT medium"). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol("PEG"). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind HKID-1, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-HKID-1 antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with HKID-1 to thereby isolateimmunoglobulin library members that bind HKID-1. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant anti-HKID-1 antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion ofHKID-1. Monoclonal antibodies directed against the antigen can beobtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as "guided selection." In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope.

First, a non-human monoclonal antibody which binds a selected antigen(epitope), e.g., an antibody which inhibits HKID-1 activity, isidentified. The heavy chain and the light chain of the non-humanantibody are cloned and used to create phage display Fab fragments. Forexample, the heavy chain gene can be cloned into a plasmid vector sothat the heavy chain can be secreted from bacteria. The light chain genecan be cloned into a phage coat protein gene so that the light chain canbe expressed on the surface of phage. A repertoire (random collection)of human light chains fused to phage is used to infect the bacteriawhich express the non-human heavy chain. The resulting progeny phagedisplay hybrid antibodies (human light chain/non-human heavy chain). Theselected antigen is used in a panning screen to select phage which bindthe selected antigen. Several rounds of selection may be required toidentify such phage. Next, human light chain genes are isolated from theselected phage which bind the selected antigen. These selected humanlight chain genes are then used to guide the selection of human heavychain genes as follows. The selected human light chain genes areinserted into vectors for expression by bacteria. Bacteria expressingthe selected human light chains are infected with a repertoire of humanheavy chains fused to phage. The resulting progeny phage display humanantibodies (human light chain/human heavy chain).

Next, the selected antigen is used in a panning screen to select phagewhich bind the selected antigen. The phage selected in this step displaya completely human antibody which recognizes the same epitope recognizedby the original selected, non-human monoclonal antibody. The genesencoding both the heavy and light chains are readily isolated and can befurther manipulated for production of human antibody. This technology isdescribed by Jespers et al. (1994, Bio/technology 12:899-903).

An anti-HKID-1 antibody (e.g., monoclonal antibody) can be used toisolate HKID-1 by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-HKID-1 antibody can facilitate thepurification of natural HKID-1 from cells and of recombinantly producedHKID-1 expressed in host cells. Moreover, an anti-HKID-1 antibody can beused to detect HKID-1 protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the HKID-1 protein. Anti-HKID-1 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵ I, ¹³¹ I, ³⁵ Sor ³ H.

IV. Recombinant Expression Vectors and Host Cells

The invention further provides vectors, preferably expression vectors,containing a nucleic acid encoding an HKID-1 protein of the presentinvention or a portion thereof.

As used herein, the term vector refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a "plasmid", which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, "operably linked" is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term "regulatory sequence" isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., HKID-1 proteins, mutant formsof HKID-1, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of HKID-1 in prokaryotic or eukaryotic cells, e.g., bacterialcells such as E. coli, insect cells (using baculovirus expressionvectors), yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, supra. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include PGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude ptrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the HKID-1 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and pPicZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, HKID-1 can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., Sf 9 cells) include the pAcseries (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to HKID-1 mRNA. Regulatory sequences operably linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (Reviews--Trendsin Genetics, Vol. 1(1) 1986).

Another aspect of the invention provides host cells into which arecombinant expression vector of the invention has been introduced. Theterms "host cell" and "recombinant host cell" are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,HKID-1 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms "transformation" and "transfection" are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (supra), andother laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Selectable markers include those which confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding HKID-1 or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) HKID-1 protein.Accordingly, the invention further provides methods for producing HKID-1protein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding HKID-1 has been introduced) in asuitable medium such that HKID-1 protein is produced. In anotherembodiment, the method further comprises isolating HKID-1 from themedium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichHKID-1-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenous HKID-1sequences have been introduced into their genome or homologousrecombinant animals in which endogenous HKID-1 sequences have beenaltered. Such animals are useful for studying the function and/oractivity of HKID-1 and for identifying and/or evaluating modulators ofHKID-1 activity. As used herein, a "transgenic animal" is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, an "homologous recombinant animal" is anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous HKID-1 gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingHKID-1-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The HKID-1cDNA sequence (e.g., that of SEQ ID NO:1 or SEQ ID NO:3) can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a nonhuman homolog of the human HKID-1 gene, such as amouse HKID-1 gene, can be isolated based on hybridization to the humanHKID-1 cDNA and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theHKID-1 transgene to direct expression of HKID-1 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the HKID-1 transgene in its genomeand/or expression of HKID-1 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding HKID-1 can further be bred to other transgenicanimals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of an HKID-1 gene (e.g., a human or anon-human homolog of the HKID-1 gene, e.g., a murine HKID-1 gene) intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the HKID-1 gene. In anembodiment, the vector is designed such that, upon homologousrecombination, the endogenous HKID-1 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a"knock out" vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous HKID-1 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous HKID-1 protein). In the homologousrecombination vector, the altered portion of the HKID-1 gene is flankedat its 5' and 3' ends by additional nucleic acid of the HKID-1 gene toallow for homologous recombination to occur between the exogenous HKID-1gene carried by the vector and an endogenous HKID-1 gene in an embryonicstem cell. The additional flanking HKID-1 nucleic acid is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5' and 3'ends) are included in the vector (see, e.g., Thomas and Capecchi (1987)Cell 51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced HKID-1 gene hashomologously recombined with the endogenous HKID-1 gene are selected(see, e.g., Li et al. (1992) Cell 69:915). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford,1987) pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomiyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of "double"transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.In brief, a cell, e.g., a somatic cell, from the transgenic animal canbe isolated and induced to exit the growth cycle and enter G_(o) phase.The quiescent cell can then be fused, e.g., through the use ofelectrical pulses, to an enucleated oocyte from an animal of the samespecies from which the quiescent cell is isolated. The reconstructedoocyte is then cultured such that it develops to morula or blastocyteand then transferred to pseudopregnant female foster animal. Theoffspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

V. Pharmaceutical Compositions

The HKID-1 nucleic acid molecules, HKID-1 proteins, and anti-HKID-1antibodies (also referred to herein as "active compounds") of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language "pharmaceutically acceptablecarrier" is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an HKID-1 protein or anti-HKID-1 antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment is sustaineduntil a desired suppression of disease symptoms occurs. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays. An exemplary dosingregimen is disclosed in WO 94/04188. The specification for the dosageunit forms of the invention are dictated by and directly dependent onthe unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologs, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology); c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). An HKID-1 protein interacts with other cellular proteinsand can thus be used as a target for developing therapeutic moleculesfor modulating HKID-1 protein in cells expressing HKID-1 protein orcells involved in the HKID-1 pathway, e.g., cells of the nervous system.The isolated nucleic acid molecules of the invention can be used toexpress HKID-1 protein (e.g., via a recombinant expression vector in ahost cell in gene therapy applications), to detect HKID-1 mRNA (e.g., ina biological sample) or a genetic lesion in an HKID-1 gene, and tomodulate HKID-1 activity. In addition, the HKID-1 proteins can be usedto screen drugs or compounds which modulate the HKID-1 activity orexpression as well as to treat disorders characterized by insufficientor excessive production of HKID-1 protein or production of HKID-1protein forms which have decreased or aberrant activity compared toHKID-1 wild type protein. In addition, the anti-HKID-1 antibodies of theinvention can be used to detect and isolate HKID-1 proteins and modulateHKID-1 activity.

This invention further provides novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

A. Screening Assays

The invention provides a method (also referred to herein as a "screeningassay") for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to HKID-1 proteins or have a stimulatory or inhibitory effecton, for example, HKID-1 expression or HKID-1 activity.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of an HKID-1protein or polypeptide or biologically active portion thereof. The testcompounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the "one-bead one-compound" library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

In an embodiment, an assay of the present invention is a cell-free assaycomprising contacting an HKID-1 protein or biologically active portionthereof with a test compound and determining the ability of the testcompound to bind to the HKID-1 protein or biologically active portionthereof. Binding of the test compound to the HKID-1 protein can bedetermined either directly or indirectly as described above. In anembodiment, the assay includes contacting the HKID-1 protein orbiologically active portion thereof with a known compound which bindsHKID-1 to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with an HKID-1 protein, wherein determining the ability of thetest compound to interact with an HKID-1 protein comprises determiningthe ability of the test compound to preferentially bind to HKID-1 orbiologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting HKID-1 protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the HKID-1 proteinor biologically active portion thereof. Determining the ability of thetest compound to modulate the activity of HKID-1 can be accomplished,for example, by determining the ability of the HKID-1 protein to bind toan HKID-1 target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of HKID-1 canbe accomplished by determining the ability of the HKID-1 protein tofurther modulate an HKID-1 target molecule. For example, thecatalytic/enzymatic activity of the target molecule on an appropriatesubstrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theHKID-1 protein or biologically active portion thereof with a knowncompound which binds HKID-1 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with an HKID-1 protein, wherein determiningthe ability of the test compound to interact with an HKID-1 proteincomprises determining the ability of the HKID-1 protein topreferentially bind to or modulate the activity of an HKID-1 targetmolecule.

Phosphoaminoacid analysis of the phosphorylated substrate can also beperformed in order to determine which residues on the HKID-1 substrateare phosphorylated. Briefly, the radiophosphorylated protein band can beexcised from the SDS gel and subjected to partial acid hydrolysis. Theproducts can then be separated by one-dimensional electrophoresis andanalyzed on, for example, a phosphoimager and compared toninhydrin-stained phosphoaminoacid standards.

In yet another embodiment of the invention, the cell free assaydetermines the ability of the HKID-1 protein to phosphorylate an HKID-1target molecule by, for example, an in vitro kinase assay. Briefly, anHKID-1 target molecule, e.g., an immunoprecipitated HKID-1 targetmolecule from a cell line expressing such a molecule, can be incubatedwith the HKID-1 protein and radioactive ATP, e.g., [gamma-³² P] ATP, ina buffer containing MgCl₂ and MnCl₂, e.g., 10 mM MgCl₂ and 5 mM MnCl₂.Following the incubation, the immunoprecipitated HKID-1 target moleculecan be separated by SDS-polyacrylamide gel electrophoresis underreducing conditions, transferred to a membrane, e.g., a PVDF membrane,and autoradiographed. The appearance of detectable bands on theautoradiograph indicates that the HKID-1 substrate has beenphosphorylated.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a soluble form of HKID-1 protein, or a biologically activeportion thereof, is contacted with a test compound and the ability ofthe test compound to bind to an HKID-1 protein determined. The cell, forexample, can be a yeast cell or a cell of mammalian origin. Determiningthe ability of the test compound to bind to the HKID-1 protein can beaccomplished, for example, by coupling the test compound with aradioisotope or enzymatic label such that binding of the test compoundto the HKID-1 protein or biologically active portion thereof can bedetermined by detecting the labeled compound in a complex. For example,test compounds can be labeled with ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Alternatively, testcompounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct. In an embodiment, the assay comprises contacting a cell whichexpresses a soluble form of HKID-1 protein, or a biologically activeportion thereof, on the cell surface with a known compound which bindsHKID-1 to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with an HKID-1 protein, wherein determining the ability of thetest compound to interact with an HKID-1 protein comprises determiningthe ability of the test compound to preferentially bind to HKID-1 or abiologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a soluble form of HKID-1 protein, or abiologically active portion thereof, with a test compound anddetermining the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the HKID-1 protein or biologicallyactive portion thereof. Determining the ability of the test compound tomodulate the activity of HKID-1 or a biologically active portion thereofcan be accomplished, for example, by determining the ability of theHKID-1 protein to bind to or interact with an HKID-1 target molecule. Asused herein, a "target molecule" is a molecule with which an HKID-1protein binds or interacts in nature, for example, a substrate moleculephosphorylated by HKID-1 protein in the interior of a cell whichexpresses an HKID-1 protein, a molecule associated with the internalsurface of a cell membrane or a cytoplasmic molecule. An HKID-1 targetmolecule can be a non-HKID-1 molecule or an HKID-1 protein orpolypeptide of the present invention. In one embodiment, an HKID-1target molecule is a component of a signal transduction pathway whichmediates transduction of a signal.

Determining the ability of the HKID-1 protein to bind to or interactwith an HKID-1 target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In an embodiment,determining the ability of the HKID-1 protein to bind to or interactwith an HKID-1 target molecule can be accomplished by determining theactivity of the target molecule. For example, the activity of the targetmolecule can be determined by detecting induction of a cellular secondmessenger of the target (e.g., intracellular Ca²⁺, diacylglycerol, IP3,etc.), detecting catalytic/enzymatic activity of the target on anappropriate substrate, detecting the induction of a reporter gene (e.g.,an HKID-1-responsive regulatory element operably linked to a nucleicacid encoding a detectable marker, e.g. luciferase), or detecting acellular response, for example, cellular differentiation, or cellproliferation.

In various formats of the assay methods of the present invention, it maybe desirable to immobilize either HKID-1 or its target molecule tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.Binding of a test compound to HKID-l, or interaction of HKID-1 with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/HKID-1fusion proteins or glutathione-S-transferase/target fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or HKID-1 protein, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of HKID-1 binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either HKID-1 orits target molecule can be immobilized utilizing conjugation of biotinand streptavidin. Biotinylated HKID-1 or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemicals). Alternatively, antibodies reactive withHKID-1 or target molecules but which do not interfere with binding ofthe HKID-1 protein to its target molecule can be derivatized to thewells of the plate, and unbound target or HKID-1 trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the HKID-1or target molecule, as well as enzyme-linked assays which rely ondetecting an enzymatic activity associated with the HKID-1 or targetmolecule.

In another embodiment, modulators of HKID-1 expression are identified ina method in which a cell is contacted with a candidate compound and theexpression of HKID-1 mRNA or protein in the cell is determined. Thelevel of expression of HKID-1 mRNA or protein in the presence of thecandidate compound is compared to the level of expression of HKID-1 mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of HKID-1 expressionbased on this comparison. For example, when expression of HKID-1 mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of HKID-1 mRNA or protein expression.Alternatively, when expression of HKID-1 mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of HKID-1 mRNA or protein expression. The level of HKID-1 mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting HKID-1 mRNA or protein.

In yet another aspect of the invention, the HKID-1 proteins can be usedas "bait proteins" in a two-hybrid assay or three hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with HKID-1 ("HKID-1-bindingproteins" or "HKID-1-bp") and modulate HKID-1 activity. SuchHKID-1-binding proteins are also likely to be involved in thepropagation of signals by the HKID-1 proteins as, for example, upstreamor downstream elements of the HKID-1 pathway. The invention alsoprovides for the use of proteins that interact with HKID-1, e.g.,two-hybrid interactors with HKID-1, as baits in two-hybrid screens andthe identification of HKID-1 interacting protein interacting proteins.HKID-1 interacting protein interacting proteins are likely to beinvolved in the HKID-1 signal transduction pathway.

This invention further provides novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Tissue Typing

The HKID-1 sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of "Dog Tags" which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the HKID-1 sequences described herein can be used toprepare two PCR primers from the 5' and 3' ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The HKID-1 sequences of the invention uniquely represent portions of thehuman genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO:3 areused, a more appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from HKID-1 sequences described herein is used togenerate a unique identification database for an individual, those samereagents can later be used to identify tissue from that individual.Using the unique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

2. Use of Partial HKID-1 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another"identification marker" (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theHKID-1 sequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO:1 having a length of at least 20 or 30bases.

The HKID-1 sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such HKID-1 probes can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., HKID-1 primers or probes canbe used to screen tissue culture for contamination (i.e., screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also provides the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningHKID-1 protein and/or nucleic acid expression as well as HKID-1activity, in the context of a biological sample (e.g., blood, serum,cells, tissue) to thereby determine whether an individual is afflictedwith a disease or disorder, or is at risk of developing a disorder,associated with aberrant HKID-1 expression or activity. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith HKID-1 protein, nucleic acid expression or activity. For example,mutations in an HKID-1 gene can be assayed in a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with HKID-1 protein, nucleic acidexpression or activity.

Another aspect of the invention provides methods for determining HKID-1protein, nucleic acid expression or HKID-1 activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as "pharmacogenomics"). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention provides monitoring the influence ofagents (e.g., drugs or other compounds) on the expression or activity ofHKID-1 in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of HKID-1 in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting HKID-1 protein or nucleic acid (e.g., mRNA, genomicDNA) that encodes HKID-1 protein such that the presence of HKID-1 isdetected in the biological sample. An agent for detecting HKID-1 mRNA orgenomic DNA can be a labeled nucleic acid probe capable of hybridizingto HKID-1 mRNA or genomic DNA. The nucleic acid probe can be, forexample, a full-length HKID-1 nucleic acid, such as the nucleic acid ofSEQ ID NO: 1 or 3, or a portion thereof, such as an oligonucleotide ofat least 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions toHKID-1 mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays of the invention are described herein.

An agent for detecting HKID-1 protein can be an antibody capable ofbinding to HKID-1 protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can beused. The term "labeled", with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term "biological sample" is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect HKID-1 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of HKID-1 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of HKID-1 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of HKID-1 genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of HKID-1 protein include introducing into a subject a labeledanti-HKID-1 antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting HKID-1 protein, mRNA, orgenomic DNA, such that the presence of HKID-1 protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofHKID-1 protein, mRNA or genomic DNA in the control sample with thepresence of HKID-1 protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of HKID-1in a biological sample (a test sample). Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping a disorder associated with aberrant expression of HKID-1(e.g., an immunological disorder). For example, the kit can comprise alabeled compound or agent capable of detecting HKID-1 protein or mRNA ina biological sample and means for determining the amount of HKID-1 inthe sample (e.g., an anti-HKID-1 antibody or an oligonucleotide probewhich binds to DNA encoding HKID-1, e.g., SEQ ID NO:1 or SEQ ID NO:3).Kits can also include instructions for observing that the tested subjectis suffering from or is at risk of developing a disorder associated withaberrant expression of HKID-1 if the amount of HKID-1 protein or mRNA isabove or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to HKID-1protein; and, optionally, (2) a second, different antibody which bindsto HKID-1 protein or the first antibody and is conjugated to adetectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labelled oligonucleotide, whichhybridizes to an HKID-1 nucleic acid sequence or (2) a pair of primersuseful for amplifying an HKID-1 nucleic acid molecule;

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of HKID-1.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant HKID-1expression or activity. For example, the assays described herein, suchas the preceding diagnostic assays or the following assays, can beutilized to identify a subject having or at risk of developing adisorder associated with HKID-1 protein, nucleic acid expression oractivity, e.g. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing such a disease ordisorder. Thus, the present invention provides a method in which a testsample is obtained from a subject and HKID-1 protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of HKID-1protein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant HKID-1expression or activity. As used herein, a "test sample" refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant HKID-1 expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with a specific agent or class of agents (e.g.,agents of a type which decrease HKID-1 activity). Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated withaberrant HKID-1 expression or activity in which a test sample isobtained and HKID-1 protein or nucleic acid is detected (e.g., whereinthe presence of HKID-1 protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant HKID-1 expression or activity).

The methods of the invention can also be used to detect genetic lesionsor mutations in an HKID-1 gene, thereby determining if a subject withthe lesioned gene is at risk for a disorder characterized by aberrantcell proliferation and/or differentiation. In embodiments, the methodsinclude detecting, in a sample of cells from the subject, the presenceor absence of a genetic lesion or mutation characterized by at least oneof an alteration affecting the integrity of a gene encoding anHKID-1-protein, or the mis-expression of the HKID-1 gene. For example,such genetic lesions or mutations can be detected by ascertaining theexistence of at least one of: 1) a deletion of one or more nucleotidesfrom an HKID-1 gene; 2) an addition of one or more nucleotides to anHKID-1 gene; 3) a substitution of one or more nucleotides of an HKID-1gene; 4) a chromosomal rearrangement of an HKID-1 gene; 5) an alterationin the level of a messenger RNA transcript of an HKID-1 gene; 6) anaberrant modification of an HKID-1 gene, such as of the methylationpattern of the genomic DNA; 7) the presence of a non-wild type splicingpattern of a messenger RNA transcript of an HKID-1 gene; 8) a non-wildtype level of an HKID-1-protein; 9) an allelic loss of an HKID-1 gene;and 10) an inappropriate post-translational modification of anHKID-1-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions inan HKID-1 gene. A biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the HKID-1-gene(see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to an HKID-1 gene under conditionssuch that hybridization and amplification of the HKID-1-gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in an HKID-1 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations in HKID-1 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al.(1996) Nature Medicine 2:753-759). For example, genetic mutations inHKID-1 can be identified in two-dimensional arrays containinglight-generated DNA probes as described in Cronin et al., supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the HKID-1 gene anddetect mutations by comparing the sequence of the sample HKID-1 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the HKID-1 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the technique of "mismatch cleavage" entailsproviding heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type HKID-1 sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. RNA/DNA duplexes can be treated with RNaseto digest mismatched regions, and DNA/DNA hybrids can be treated with S1nuclease to digest mismatched regions. In other embodiments, eitherDNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmiumtetroxide and with piperidine in order to digest mismatched regions.After digestion of the mismatched regions, the resulting material isthen separated by size on denaturing polyacrylamide gels to determinethe site of mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad.Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. Inan embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called "DNA mismatch repair" enzymes) in defined systems fordetecting and mapping point mutations in HKID-1 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on an HKID-1sequence, e.g., a wild-type HKID-1 sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, e.g., U.S.Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in HKID-1 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). Single-stranded DNA fragments of sample and control HKID-1nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,and the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In an embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of oneprimer where, under appropriate conditions, mismatch can prevent orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3'end of the 5' sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving an HKID-1 gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which HKID-1 is expressed may be utilized in theprognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onHKID-1 activity (e.g., HKID-1 gene expression) as identified by ascreening assay described herein can be administered to individuals totreat (prophylactically or therapeutically) disorders (e.g., disordersinvolving cells or tissues in which HKID-1 is expressed, such as cellsof the nervous system) associated with aberrant HKID-1 activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of HKID-1 protein,expression of HKID-1 nucleic acid, or mutation content of HKID-1 genesin an individual can be determined to thereby select appropriateagent(s) for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as "altered drugaction." Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as "altered drug metabolism".These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2Cl9) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of HKID-1 protein, expression of HKID-1 nucleic acid,or mutation content of HKID-1 genes in an individual can be determinedto thereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual. In addition, pharmacogenetic studies can beused to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith an HKID-1 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of HKID-1 (e.g., the ability to modulate aberrantcell proliferation and/or differentiation) can be applied not only inbasic drug screening, but also in clinical trials. For example, theeffectiveness of an agent, as determined by a screening assay asdescribed herein, to increase HKID-1 gene expression, protein levels orprotein activity, can be monitored in clinical trials of subjectsexhibiting decreased HKID-1 gene expression, protein levels, or proteinactivity. Alternatively, the effectiveness of an agent, as determined bya screening assay, to decrease HKID-1 gene expression, protein levels orprotein activity, can be monitored in clinical trials of subjectsexhibiting increased HKID-1 gene expression, protein levels, or proteinactivity. In such clinical trials, HKID-1 expression or activity andpreferably, that of other genes that have been implicated in forexample, a cellular proliferation disorder, can be used as a marker ofthe immune responsiveness of a particular cell.

For example, and not by way of limitation, genes, including HKID-1, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates HKID-1 activity (e.g., as identifiedin a screening assay described herein) can be identified. Thus, to studythe effect of agents on cellular proliferation disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of HKID-1 and other genes implicated in thedisorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, by hybridization to a multiple tissue expression arrayas described in Example 2, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of HKID-1 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In an embodiment, the present invention provides a method for monitoringthe effectiveness of treatment of a subject with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate identified by the screeningassays described herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of an HKID-1 protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the HKID-1 protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the HKID-1 protein, mRNA, or genomic DNA inthe pre-administration sample with the HKID-1 protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of HKID-1 to higher levels than detected, i.e.,to increase the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of HKID-1 to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant HKID-1 expression oractivity. Such disorders include

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant HKID-1expression or activity, by administering to the subject an agent whichmodulates HKID-1 expression or at least one HKID-1 activity. Subjects atrisk for a disease which is caused or contributed to by aberrant HKID-1expression or can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the HKID-1 aberrancy, such that a disease or disorderis prevented or, alternatively, delayed in its progression. Depending onthe type of HKID-1 aberrancy, for example, an HKID-1 agonist or HKID-1antagonist agent can be used for treating the subject. The appropriateagent can be determined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention provides methods of modulating HKID-1expression or activity for therapeutic purposes. The modulatory methodof the invention involves contacting a cell with an agent that modulatesone or more of the activities of HKID-1 protein activity associated withthe cell. An agent that modulates HKID-1 protein activity can be anagent as described herein, such as a small molecule, e.g., a smallmolecule that modulates the protein kinase activity of HKID-1, a nucleicacid or a protein, a naturally-occurring cognate ligand of an HKID-1protein, a peptide, or an HKID-1 peptidomimetic. In one embodiment, theagent stimulates one or more of the biological activities of HKID-1protein. Examples of such stimulatory agents include small moleculesthat stimulate one or more activities of HKID-1, e.g., the HKID-1protein kinase activity, active HKID-1 protein and a nucleic acidmolecule encoding HKID-1 that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more of the biologicalactivities of HKID-1 protein. Examples of such inhibitory agents includea small molecule that inhibits one or more HKID-1 activities e.g.,HKID-1 protein kinase activity, antisense HKID-1 nucleic acid moleculesand anti-HKID-1 antibodies. These modulatory methods can be performed invitro (e.g., by culturing the cell with the agent) or, alternatively, invivo (e.g, by administering the agent to a subject). As such, thepresent invention provides methods of treating an individual afflictedwith a disease or disorder characterized by aberrant expression oractivity of an HKID-1 protein or nucleic acid molecule. The presentinvention also provides methods of treating an individual afflicted witha disease or disorder that can be treated by modulating the activity ofHKID-1 an HKID-1 protein or nucleic acid molecule. In one embodiment,the method involves administering an agent, e.g., a small molecule,(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) HKID-1 expression or activity.

Stimulation of HKID-1 activity is desirable in situations in whichHKID-1 is abnormally downregulated and/or in which increased HKID-1activity is likely to have a beneficial effect. Conversely, inhibitionof HKID-1 activity is desirable in situations in which HKID-1 isabnormally upregulated and/or in which decreased HKID-1 activity islikely to have a beneficial effect.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES Example 1 Determination of the Nucleotide Sequence of HKID-1

Human HKID-1 cDNAs iolated from cDNA libraries constructed in standardcloning vectors were sequenced. The cDNA sequences were assembled into acontig and the HKID-1 sequence was determined from the consensussequence of this contig. Analysis of the contig revealed anapproximately 2126 kb HKID-1 cDNA sequence with a 978 base pair openreading frame predicted to encode a novel 326 amino acid protein.

Example 2 Distribution of HKID-1 mRNA in Human Tissues

HKID-1 mRNA expression was analyzed by hybridizing a radioactivelylabeled HKID-1-specific DNA probe to human poly A+ RNA arrayed on anylon membrane (the Human Multiple Tissue Expression (MTE) Array,Clontech; Palo Alto, Calif.). Poly A+ RNAs from the following humantissues and cell lines are present on the MTE Array, whole brain,cerebral cortex, frontal lobe, parietal lobe, occipital lobe, temporallobe, paracentral gyrus of cerebral cortex, pons, left cerebellum, rightcerebellum, corpus callosum, amygdala, caudate nucleus, hippocampus,medulla oblongata, putamen, substantia nigra, accumbens nucleus,thalamus, pituitary gland, spinal cord, heart, aorta, left atrium, rightatrium, left ventricle, right ventricle, interventricular septum, apexof the heart, esophagus, stomach, duodenum, jejunum, ileum, ilocecum,appendix, ascending colon, transverse colon, descending colon, rectum,kidney, skeletal muscle, spleen, thymus, peripheral blood leukocyte,lymph node, bone marrow, trachea, lung, placenta, bladder, uterus,prostate, testis, ovary, liver, pancreas, adrenal gland, thyroid gland,salivary gland, mammary gland, HL-60 leukemia cell line, S3 HeLa cellline, K-562 leukemia cell line, MOLT-4 leukemia cell line, RajiBurkitt's lymphoma cell line, Daudi Burkitt's lymphoma cell line, SW480colorectal adeno-carcinoma cell line, A549 lung carcinoma cell line,fetal brain, fetal heart, fetal kidney, fetal liver, fetal spleen, fetalthymus, fetal lung.

In more detail, a portion of the HKID-1 cDNA was synthesized using PCRfor use as a hybridization probe. The HKID-1 specific DNA wasradioactively labeled with 32P-dCTP using the Prime-It kit (Stratagene;La Jolla, Calif.) according to the instructions of the supplier. The MTEarray filter was probed with the radiolabeled HKID-1 specific DNA probein ExpressHyb hybridization solution (Clontech) and washed at highstringency according to the manufacturer's recommendations. Thesestudies revealed that HKID-1 mRNA is expressed in all tissues containedin the MTE array. The highest expression in adult tissues was detectedin placenta then trachea then lung then peripheral blood leukocytes thenheart. In fetal tissues, the highest expression was detected in lungthen heart then kidney then spleen. Low expression of HKID-1 mRNA wasdetected in all tissues analyzed. HKID-1 mRNA expression was weakoverall in both adult and fetal brain except in adult substantia nigraand adult pituitary gland in which HKID-1 mRNA levels were moderate.

Example 3 Characterization of HKID-1 Protein

In this example, the predicted amino acid sequence of human HKID-1protein was compared to amino acid sequences of known motifs and/ordomains present in proteins and to the polypeptide sequences of knownproteins. Polypeptide domains and/or motifs present in HKID-1 wereidentified as were proteins with significant amino acid similarities toHKID-1. In addition, the molecular weight of the human HKID-1 proteinwas predicted.

The human HKID-1 nucleotide sequence (FIG. 1; SEQ ID NO:1), identifiedas described above, encodes a 326 amino acid protein (FIG. 1; SEQ IDNO:2). HKID-1 has a predicted MW of about 35.86 kDa, not includingpost-translational modifications. The HKID-1 polypeptide sequence of SEQID NO:2 was used to query the PROSITE database of protein patterns andto query a library of Hidden Markov Models (HMMs) which can recognizecommon protein domains and families. The search of the PROSITE databaserevealed the presence of one cAMP- and cGMP-dependent protein kinasephosphorylation site (PS00004) from amino acids 260-263 of SEQ ID NO:2;three protein kinase C phosphorylation sites (PS00005) from amino acids137-139, 275-277, and 279-281, of SEQ ID NO:2; three casein kinase IIphosphorylation sites (PS00006) from amino acids 202-205, 211-214, and321-324, of SEQ ID NO:2; one tyrosine kinase phosphorylation site(PS00007; SEQ ID NO:14) from amino acid 33-40, of SEQ ID NO:2 sevenN-myristoylation sites (PS00008) from amino acids 43-48, 49-54, 57-62,63-68, 80-85, 98-103, and 295-300 of SEQ ID NO:2; one protein kinaseATP-binding region signature (PS00107) from amino acid 46-54, of SEQ IDNO:2; one serine/threonine protein kinase active site signature(PS00108) from amino acid 166-178, of SEQ ID NO:2. The search of the HMMdatabase revealed the presence of one eukaryotic protein kinase domain(PF00069; SEQ ID NO:4) from amino acid 40-293, of SEQ ID NO:2; with ascore of 262.4 and E value of 5.9×10 75 (see FIG. 2). For generalinformation regarding PFAM identifiers, PS prefix and PF prefix motifidentification numbers, refer to Sonnhammer et al. (1997) Protein28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html.

The HKID-1 polypeptide sequence of SEQ ID NO:2 was used to query thePROTOT database of protein sequences using the BLASTP program with theBLOSUM62 matrix and a protein word length of 3. The five most closelyrelated proteins to HKID-1 identified by this BLASTP analysis arelisted: HKID-1 was found to be 95% identical over 326 amino acids to ratKID-1 (AF086624; SEQ ID NO:5) with a score of 1646, 77% identical toXenopus laevis (frog) PIM-1 (Q91822; SEQ ID NO:6) with a score of 922,similar to murine PIM-1 (P06803; SEQ ID NO:7) with a score of 873,similar to rat PIM-1 (P26794; SEQ ID NO:8) with a score of 884, andsimilar to human PIM-1 (P11309; SEQ ID NO:9) with a score of 883.

FIG. 4 shows an alignment, carried out with the MegAlign program of theDNASTAR sequence analysis package using the J. Hein method with a PAM250residue weight table, of the HKID-1 polypeptide sequence of SEQ ID NO:2and the just listed five closest HKID-1 relatives identified by BLASTPanalysis. Table 1 shows both the percent polypeptide sequence similarityand the percent polypeptide sequence divergence between HKID-1 and itsfive closest relatives identified by BLASTP analysis as well as thepercent polypeptide sequence similarity and the percent polypeptidesequence divergence between said HKID-1 relatives and each other.Sequence pair distances were carried out with the MegAlign program ofthe DNASTAR sequence analysis package using the J. Hein method with aPAM250 residue weight table. These analyses indicate that HKID-1 is thespecies ortholog of rat KID-1 (Feldman, J. D. et al. (1998). J. Biol.Chem. 273:16535-16543) and frog PIM-1 because HKID-1 is more closelyrelated to these two proteins than to PIM-1 proteins. It has beenreported that frog PIM-1 and rat KID-1 are species orthologs (Feldman,J. D. et al. (1998). J. Biol. Chem. 273:16535-16543). HKID-1 is aparalog of human PIM-1, murine PIM-1, and rat PIM-1. HKID-1 plays someor all of the roles in human that its species orthologs, rat KID-1 andfrog PIM-1, play in the species from which they originate.

The rat KID-1, frog PIM-1, and human and murine PIM-1 are all known tohave serine/threonine protein kinase activity in in vitrophosphorylation assays. The high polypeptide sequence similarity betweenHKID-1 and rat KID-1, frog PIM-1, and human and murine PIM-1, HKID-1demonstrates that HKID-1 is a serine/threonine protein kinase.

Rat KID-1 is described in Feldman, J. D. et al. (1998). J. Biol. Chem.273:16535-16543. Rat KID-1 is induced in specific regions of thehippocampus and cortex in response to kainic acid and electroconvulsiveshock suggesting that rat KID-1 is involved in neuronal function,synaptic plasticity, learning, and memory as well as kainic acidseizures and some nervous system-related diseases such as seizures andepilepsy. Because HKID-1 is the species ortholog of rat KID-1, HKID-1 isinvolved in some or all of the processes and diseases in which rat KID-1is involved. In addition, the HKID-1 paralogs, the PIM-1 proteins, areproto-oncogenes. Consequently, it is possible that HKID-1 is involved incell growth regulation, cancer, and related pathways and diseases.

                                      TABLE 1                                     __________________________________________________________________________            frog      human                                                                              murine                                                                             rat  rat                                            PIM-1 HKID-1 PIM-1 PIM-1 KID-1 PIM-1                                        __________________________________________________________________________    frog PIM-1                                                                            ***  77.5 65.5 66.1 77.2 65.5 frog PIM-1                                HKID-1 26.8 *** 68.7 68.4 95.4 69.0 HKID-1                                    human PIM-1 46.0 40.5 *** 93.9 68.7 97.1 human PIM-1                          murine PIM-1 44.9 41.0  6.3 *** 68.4 94.3 murine PIM-1                        rat KID-1 27.3  4.7 40.5 41.0 *** 68.7 rat KID-1                              rat PIM-1 46.0 39.9  2.9  6.0 40.5 *** rat PIM-1                            __________________________________________________________________________

Table Legend: Pair distances of HKID-1 and the five most closely relatedproteins identified in a BLASTP analysis. Percent similarity is shown inthe upper triangular quadrant and percent divergence in shown in thelower triangular quadrant. Sequence pair distances were carried out withthe MegAlign program of the DNASTAR sequence analysis package using theJ. Hein method with a PAM250 residue weight table.

Example 4 Preparation of HKID-1 Fusion Proteins

Recombinant HKID-1 can be produced in a variety of expression systems.For example, the mature HKID-1 peptide can be expressed as a recombinantglutathione-S-transferase (GST) fusion protein in E. coli and the fusionprotein can be isolated and characterized. Specifically, as describedabove, HKID-1 can be fused to GST and this fusion protein can beexpressed in E. coli strain PEB199. Expression of the GST-HKID-1 fusionprotein in PEB199 can be induced with IPTG. The recombinant fusionprotein can be purified from crude bacterial lysates of the inducedPEB199 strain by affinity chromatography on glutathione beads.

Example 5 Identification of the chromosomal location of HKID-1

To determine the chromosomal location of HKID-1, the HKID-1 nucleotidesequence of SEQ ID NO:1 was used to query, using the BLASTN program(Altschul S. F. et al, (1990) J. Mol. Biol. 215: 403-410.) with a wordlength of 12 and using the BLOSUM62 scoring matrix, a database of humannucleotide sequences originating from nucleotide molecules that havebeen mapped to the human genome. The WI-11798 nucleotide sequence wasfound to contain HKID-1 sequences establishing that WI-11798 and HKID-1map to the same chromosomal location, chromosome 22 between the D22S1169and D22S₋₋ qter markers, 196.70 centiRays from the top of the chromosome22 linkage group.

Example 6 Tissue Distribution of HKID-1 mRNA by Large-ScaleTissue-Specific Library Sequencing

Standard molecular biology methods (Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) were used to construct cDNA libraries inplasmid vectors from multiple human tissues. Individual cDNA clones fromeach library were isolated and sequenced and their nucleotide sequenceswere input into a database. The HKID-1 nucleotide sequence of SEQ IDNO:1 was used to query the tissue-specific library cDNA clone nucleotidesequence database using the BLASTN program (Altschul S. F. et al, (1990)J. Mol. Biol. 215: 403-410.) with a word length of 12 and using theBLOSUM62 scoring matrix. Nucleotide sequences identical to portions ofthe HKID-1 nucleotide sequence of SEQ ID NO:1 were found in cDNAlibraries originating from human skin, kidney, lung, heart, thymus,endothelial cells, prostate, uterus, lymph node, neuron, placenta,T-cell, breast and muscle. This result indicates that the HKID-1 mRNA,or fragments thereof, is expressed in the listed tissues, although it isnot possible to draw any conclusion about the expression level of HKID-1mRNA in said tissues. In addition, the fact that HKID-1-identicalsequences were not detected in libraries originating from other tissuesdoes not mean that the HKID-1 mRNA is not expressed in those tissues.HKID-1 nucleic acid sequences, fragments thereof, proteins encoded bythese sequences, and fragments thereof as well as modulators of HKID-1gene or protein activity may be useful for diagnosing or treatingdiseases that involve the tissues in which the HKID-1 mRNA is expressed.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 11                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 2126                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - ggcgctccgc ctgctgcgcg tctacgcggt ccccgcgggc cttccgggcc ca -            #ctgcgccg     60                                                                 - - cgcggaccgc ctcgggctcg gacggccggt gtccccggcg cgccgctcgc cc -            #ggatcggc    120                                                                 - - cgcggcttcg gcgcctgggg ctcggggctc cggggaggcc gtcgcccgcg at -            #gctgctct    180                                                                 - - ccaagttcgg ctccctggcg cacctctgcg ggcccggcgg cgtggaccac ct -            #cccggtga    240                                                                 - - agatcctgca gccagccaag gcggacaagg agagcttcga gaaggcgtac ca -            #ggtgggcg    300                                                                 - - ccgtgctggg tagcggcggc ttcggcacgg tctacgcggg tagccgcatc gc -            #cgacgggc    360                                                                 - - tcccggtggc tgtgaagcac gtggtgaagg agcgggtgac cgagtggggc ag -            #cctgggcg    420                                                                 - - gcgcgaccgt gcccctggag gtggtgctgc tgcgcaaggt gggcgcggcg gg -            #cggcgcgc    480                                                                 - - gcggcgtcat ccgcctgctg gactggttcg agcggcccga cggcttcctg ct -            #ggtgctgg    540                                                                 - - agcggcccga gccggcgcag gacctcttcg actttatcac ggagcgcggc gc -            #cctggacg    600                                                                 - - agccgctggc gcgccgcttc ttcgcgcagg tgctggccgc cgtgcgccac tg -            #ccacagct    660                                                                 - - gcggggtcgt gcaccgcgac attaaggacg aaaatctgct tgtggacctg cg -            #ctccggag    720                                                                 - - agctcaagct catcgacttc ggttcgggtg cgctgctcaa ggacacggtc ta -            #caccgact    780                                                                 - - tcgacggcac ccgagtgtac agccccccgg agtggatccg ctaccaccgc ta -            #ccacgggc    840                                                                 - - gctcggccac cgtgtggtcg ctgggcgtgc ttctctacga tatggtgtgt gg -            #ggacatcc    900                                                                 - - ccttcgagca ggacgaggag atcctccgag gccgcctgct cttccggagg ag -            #ggtctctc    960                                                                 - - cagagtgcca gcagctgatc cggtggtgcc tgtccctgcg gccctcagag cg -            #gccgtcgc   1020                                                                 - - tggatcagat tgcggcccat ccctggatgc tgggggctga cgggggcgcc cc -            #ggagagct   1080                                                                 - - gtgacctgcg gctgtgcacc ctcgaccctg atgacgtggc cagcaccacg tc -            #cagcagcg   1140                                                                 - - agagcttgtg aggagctgca cctgactggg agctagggga ccacctgcct tg -            #gccagacc   1200                                                                 - - tgggacgccc ccagaccctg actttttcct gcgtgggccg tctcctcctg cg -            #gaagcagt   1260                                                                 - - gacctctgac ccctggtgac cttcgctttg agtgcctttt gaacgctggt cc -            #cgcgggac   1320                                                                 - - ttggttttct caagctctgt ctgtccaaag acgctccggt cgaggtcccg cc -            #tgccctgg   1380                                                                 - - gtggatactt gaaccccaga cgcccctctg tgctgctgtg tccggaggcg gc -            #cttcccat   1440                                                                 - - ctgcctgccc acccggagct ctttccgccg gcgcagggtc ccaagcccac ct -            #cccgccct   1500                                                                 - - cagtcctgcg gtgtgcgtct gggcacgtcc tgcacacaca atgcaagtcc tg -            #gcytccgc   1560                                                                 - - gcccgcccgc ccacgcgagc cgtacccgcc gccaactctg ttatttatgg tg -            #tgaccccc   1620                                                                 - - tggaggtgcc ctcggcccac cggggctatt tattgtttaa tttatttgtt ga -            #ggttattt   1680                                                                 - - cctctgagca gtctgcctct cccaagcccc aggggacagt ggggaggcag gg -            #gagggggt   1740                                                                 - - ggctgtggtc cagggacccc aggccctgat tcctgtgcct ggcgtctgtc ct -            #ggccccgc   1800                                                                 - - ctgtcagaag atgaacatgt atagtggcta acttaagggg agtgggtgac cc -            #tgacactt   1860                                                                 - - ccaggcactg tgcccagggt ttgggtttta aattattgac tttgtacagt ct -            #gcttgtgg   1920                                                                 - - gctctgaaag ctggggtggg gccagagcct gagcgtttaa tttattcagt ac -            #ctgtgttt   1980                                                                 - - gtgtgaatgc ggtgtgtgca ggcatcgcag atgggggttc tttcagttca aa -            #agtgagat   2040                                                                 - - gtctggagat catatttttt tatacaggta tttcaattaa aatgtttttg ta -            #catagaaa   2100                                                                 - - aaaaaaaaaa aaaaaaaaaa gggcgg          - #                  - #                2126                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 326                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - Met Leu Leu Ser Lys Phe Gly Ser Leu Ala Hi - #s Leu Cys Gly Pro Gly        1               5 - #                 10 - #                 15              - - Gly Val Asp His Leu Pro Val Lys Ile Leu Gl - #n Pro Ala Lys Ala Asp                   20     - #             25     - #             30                  - - Lys Glu Ser Phe Glu Lys Ala Tyr Gln Val Gl - #y Ala Val Leu Gly Ser               35         - #         40         - #         45                      - - Gly Gly Phe Gly Thr Val Tyr Ala Gly Ser Ar - #g Ile Ala Asp Gly Leu           50             - #     55             - #     60                          - - Pro Val Ala Val Lys His Val Val Lys Glu Ar - #g Val Thr Glu Trp Gly       65                 - # 70                 - # 75                 - # 80       - - Ser Leu Gly Gly Ala Thr Val Pro Leu Glu Va - #l Val Leu Leu Arg Lys                       85 - #                 90 - #                 95              - - Val Gly Ala Ala Gly Gly Ala Arg Gly Val Il - #e Arg Leu Leu Asp Trp                  100      - #           105      - #           110                  - - Phe Glu Arg Pro Asp Gly Phe Leu Leu Val Le - #u Glu Arg Pro Glu Pro              115          - #       120          - #       125                      - - Ala Gln Asp Leu Phe Asp Phe Ile Thr Glu Ar - #g Gly Ala Leu Asp Glu          130              - #   135              - #   140                          - - Pro Leu Ala Arg Arg Phe Phe Ala Gln Val Le - #u Ala Ala Val Arg His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys His Ser Cys Gly Val Val His Arg Asp Il - #e Lys Asp Glu Asn        Leu                                                                                             165  - #               170  - #               175             - - Leu Val Asp Leu Arg Ser Gly Glu Leu Lys Le - #u Ile Asp Phe Gly Ser                  180      - #           185      - #           190                  - - Gly Ala Leu Leu Lys Asp Thr Val Tyr Thr As - #p Phe Asp Gly Thr Arg              195          - #       200          - #       205                      - - Val Tyr Ser Pro Pro Glu Trp Ile Arg Tyr Hi - #s Arg Tyr His Gly Arg          210              - #   215              - #   220                          - - Ser Ala Thr Val Trp Ser Leu Gly Val Leu Le - #u Tyr Asp Met Val Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Asp Ile Pro Phe Glu Gln Asp Glu Glu Il - #e Leu Arg Gly Arg        Leu                                                                                             245  - #               250  - #               255             - - Leu Phe Arg Arg Arg Val Ser Pro Glu Cys Gl - #n Gln Leu Ile Arg Trp                  260      - #           265      - #           270                  - - Cys Leu Ser Leu Arg Pro Ser Glu Arg Pro Se - #r Leu Asp Gln Ile Ala              275          - #       280          - #       285                      - - Ala His Pro Trp Met Leu Gly Ala Asp Gly Gl - #y Ala Pro Glu Ser Cys          290              - #   295              - #   300                          - - Asp Leu Arg Leu Cys Thr Leu Asp Pro Asp As - #p Val Ala Ser Thr Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Ser Ser Glu Ser Leu                                                                  325                                                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 978                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - atgctgctct ccaagttcgg ctccctggcg cacctctgcg ggcccggcgg cg -            #tggaccac     60                                                                 - - ctcccggtga agatcctgca gccagccaag gcggacaagg agagcttcga ga -            #aggcgtac    120                                                                 - - caggtgggcg ccgtgctggg tagcggcggc ttcggcacgg tctacgcggg ta -            #gccgcatc    180                                                                 - - gccgacgggc tcccggtggc tgtgaagcac gtggtgaagg agcgggtgac cg -            #agtggggc    240                                                                 - - agcctgggcg gcgcgaccgt gcccctggag gtggtgctgc tgcgcaaggt gg -            #gcgcggcg    300                                                                 - - ggcggcgcgc gcggcgtcat ccgcctgctg gactggttcg agcggcccga cg -            #gcttcctg    360                                                                 - - ctggtgctgg agcggcccga gccggcgcag gacctcttcg actttatcac gg -            #agcgcggc    420                                                                 - - gccctggacg agccgctggc gcgccgcttc ttcgcgcagg tgctggccgc cg -            #tgcgccac    480                                                                 - - tgccacagct gcggggtcgt gcaccgcgac attaaggacg aaaatctgct tg -            #tggacctg    540                                                                 - - cgctccggag agctcaagct catcgacttc ggttcgggtg cgctgctcaa gg -            #acacggtc    600                                                                 - - tacaccgact tcgacggcac ccgagtgtac agccccccgg agtggatccg ct -            #accaccgc    660                                                                 - - taccacgggc gctcggccac cgtgtggtcg ctgggcgtgc ttctctacga ta -            #tggtgtgt    720                                                                 - - ggggacatcc ccttcgagca ggacgaggag atcctccgag gccgcctgct ct -            #tccggagg    780                                                                 - - agggtctctc cagagtgcca gcagctgatc cggtggtgcc tgtccctgcg gc -            #cctcagag    840                                                                 - - cggccgtcgc tggatcagat tgcggcccat ccctggatgc tgggggctga cg -            #ggggcgcc    900                                                                 - - ccggagagct gtgacctgcg gctgtgcacc ctcgaccctg atgacgtggc ca -            #gcaccacg    960                                                                 - - tccagcagcg agagcttg             - #                  - #                      - # 978                                                                  - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 254                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: eukaryoti           protein kinase domain                                                    - - <400> SEQUENCE: 4                                                         - - Tyr Gln Val Gly Ala Val Leu Gly Ser Gly Gl - #y Phe Gly Thr Val Tyr        1               5 - #                 10 - #                 15              - - Ala Gly Ser Arg Ile Ala Asp Gly Leu Pro Va - #l Ala Val Lys His Val                   20     - #             25     - #             30                  - - Val Lys Glu Arg Val Thr Glu Trp Gly Ser Le - #u Gly Gly Ala Thr Val               35         - #         40         - #         45                      - - Pro Leu Glu Val Val Leu Leu Arg Lys Val Gl - #y Ala Ala Gly Gly Ala           50             - #     55             - #     60                          - - Arg Gly Val Ile Arg Leu Leu Asp Trp Phe Gl - #u Arg Pro Asp Gly Phe       65                 - # 70                 - # 75                 - # 80       - - Leu Leu Val Leu Glu Arg Pro Glu Pro Ala Gl - #n Asp Leu Phe Asp Phe                       85 - #                 90 - #                 95              - - Ile Thr Glu Arg Gly Ala Leu Asp Glu Pro Le - #u Ala Arg Arg Phe Phe                  100      - #           105      - #           110                  - - Ala Gln Val Leu Ala Ala Val Arg His Cys Hi - #s Ser Cys Gly Val Val              115          - #       120          - #       125                      - - His Arg Asp Ile Lys Asp Glu Asn Leu Leu Va - #l Asp Leu Arg Ser Gly          130              - #   135              - #   140                          - - Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Al - #a Leu Leu Lys Asp Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Ty - #r Ser Pro Pro Glu        Trp                                                                                             165  - #               170  - #               175             - - Ile Arg Tyr His Arg Tyr His Gly Arg Ser Al - #a Thr Val Trp Ser Leu                  180      - #           185      - #           190                  - - Gly Val Leu Leu Tyr Asp Met Val Cys Gly As - #p Ile Pro Phe Glu Gln              195          - #       200          - #       205                      - - Asp Glu Glu Ile Leu Arg Gly Arg Leu Leu Ph - #e Arg Arg Arg Val Ser          210              - #   215              - #   220                          - - Pro Glu Cys Gln Gln Leu Ile Arg Trp Cys Le - #u Ser Leu Arg Pro Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Arg Pro Ser Leu Asp Gln Ile Ala Ala Hi - #s Pro Trp Met                             245  - #               250                                     - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 455                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Rattus norvegicus                                              - - <400> SEQUENCE: 5                                                         - - Met Pro Lys Leu His Gln Pro Leu Val Asn Ar - #g Gln Gly Ala Ser Gly        1               5 - #                 10 - #                 15              - - Phe Pro Ser Thr Thr Leu Pro Asp Ser Lys Gl - #n Pro His Arg Lys Val                   20     - #             25     - #             30                  - - Ser Leu Gly Arg Lys Glu Ala Glu Leu Gln Al - #a Ala Pro Pro Pro Arg               35         - #         40         - #         45                      - - Arg Asp Thr Cys Leu Arg Gly Pro Lys Pro Ar - #g Gly Glu Ala Ala Gly           50             - #     55             - #     60                          - - Ala Cys Glu Pro Leu Gly Gln Leu Pro Ser Th - #r Gly Phe Arg Ala Ala       65                 - # 70                 - # 75                 - # 80       - - Thr Gly Gln Leu Arg Arg Ala Ala Ala Pro Le - #u Ser Ala Arg Pro Arg                       85 - #                 90 - #                 95              - - Gly Arg Gly Ile Arg Arg Ala Val Cys Gly Gl - #n Glu Asp Arg Pro Pro                  100      - #           105      - #           110                  - - Ala Ser Val Pro Asp Gly Ser Glu Ala Ala Pr - #o His Ala Arg Pro Pro              115          - #       120          - #       125                      - - Ala Met Leu Leu Ser Lys Phe Gly Ser Leu Al - #a His Leu Cys Gly Pro          130              - #   135              - #   140                          - - Gly Gly Val Asp His Leu Pro Val Lys Ile Le - #u Gln Pro Ala Lys Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Lys Glu Ser Phe Glu Lys Val Tyr Gln Va - #l Gly Ala Val Leu        Gly                                                                                             165  - #               170  - #               175             - - Ser Gly Gly Phe Gly Thr Val Tyr Ala Gly Se - #r Arg Ile Ala Asp Gly                  180      - #           185      - #           190                  - - Leu Pro Val Ala Val Lys His Val Val Lys Gl - #u Arg Val Thr Glu Trp              195          - #       200          - #       205                      - - Gly Ser Leu Gly Gly Met Ala Val Pro Leu Gl - #u Val Val Leu Leu Arg          210              - #   215              - #   220                          - - Lys Val Gly Ala Ala Gly Gly Ala Arg Gly Va - #l Ile Arg Leu Leu Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Trp Phe Glu Arg Pro Asp Gly Phe Leu Leu Va - #l Leu Glu Arg Pro        Glu                                                                                             245  - #               250  - #               255             - - Pro Ala Gln Asp Leu Phe Asp Phe Ile Thr Gl - #u Arg Gly Ala Leu Asp                  260      - #           265      - #           270                  - - Glu Pro Leu Ala Arg Arg Phe Phe Ala Gln Va - #l Leu Ala Ala Val Arg              275          - #       280          - #       285                      - - His Cys His Asn Cys Gly Val Val His Arg As - #p Ile Lys Asp Glu Asn          290              - #   295              - #   300                          - - Leu Leu Val Asp Leu Arg Ser Gly Glu Leu Ly - #s Leu Ile Asp Phe Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Gly Ala Val Leu Lys Asp Thr Val Tyr Th - #r Asp Phe Asp Gly        Thr                                                                                             325  - #               330  - #               335             - - Arg Val Tyr Ser Pro Pro Glu Trp Ile Arg Ty - #r His Arg Tyr His Gly                  340      - #           345      - #           350                  - - Arg Ser Ala Thr Val Trp Ser Leu Gly Val Le - #u Leu Tyr Asp Met Val              355          - #       360          - #       365                      - - Cys Gly Asp Ile Pro Phe Glu Gln Asp Glu Gl - #u Ile Leu Arg Gly Arg          370              - #   375              - #   380                          - - Leu Phe Phe Arg Arg Arg Val Ser Pro Glu Cy - #s Gln Gln Leu Ile Glu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Trp Cys Leu Ser Leu Arg Pro Ser Glu Arg Pr - #o Ser Leu Asp Gln        Ile                                                                                             405  - #               410  - #               415             - - Ala Ala His Pro Trp Met Leu Gly Thr Glu Gl - #y Ser Val Pro Glu Asn                  420      - #           425      - #           430                  - - Cys Asp Leu Arg Leu Cys Ala Leu Asp Thr As - #p Asp Gly Ala Ser Thr              435          - #       440          - #       445                      - - Thr Ser Ser Ser Glu Ser Leu                                                  450              - #   455                                                 - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 323                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Xenopus laevis                                                 - - <400> SEQUENCE: 6                                                         - - Met Leu Leu Ser Lys Phe Gly Ser Leu Ala Hi - #s Ile Cys Asn Pro Ser        1               5 - #                 10 - #                 15              - - Asn Met Glu His Leu Pro Val Lys Ile Leu Gl - #n Pro Val Lys Val Asp                   20     - #             25     - #             30                  - - Lys Glu Pro Phe Glu Lys Val Tyr Gln Val Gl - #y Ser Val Val Ala Ser               35         - #         40         - #         45                      - - Gly Gly Phe Gly Thr Val Tyr Ser Asp Ser Ar - #g Ile Ala Asp Gly Gln           50             - #     55             - #     60                          - - Pro Val Ala Val Lys His Val Ala Lys Glu Ar - #g Val Thr Glu Trp Gly       65                 - # 70                 - # 75                 - # 80       - - Thr Leu Asn Gly Val Met Val Pro Leu Glu Il - #e Val Leu Leu Lys Lys                       85 - #                 90 - #                 95              - - Val Pro Thr Ala Phe Arg Gly Val Ile Asn Le - #u Leu Asp Trp Tyr Glu                  100      - #           105      - #           110                  - - Arg Pro Asp Ala Phe Leu Ile Val Met Glu Ar - #g Pro Glu Pro Val Lys              115          - #       120          - #       125                      - - Asp Leu Phe Asp Tyr Ile Thr Glu Lys Gly Pr - #o Leu Asp Glu Asp Thr          130              - #   135              - #   140                          - - Ala Arg Gly Phe Phe Arg Gln Val Leu Glu Al - #a Val Arg His Cys Tyr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asn Cys Gly Val Val His Arg Asp Ile Lys As - #p Glu Asn Leu Leu        Val                                                                                             165  - #               170  - #               175             - - Asp Thr Arg Asn Gly Glu Leu Lys Leu Ile As - #p Phe Gly Ser Gly Ala                  180      - #           185      - #           190                  - - Leu Leu Lys Asp Thr Val Tyr Thr Asp Phe As - #p Gly Thr Arg Val Tyr              195          - #       200          - #       205                      - - Ser Pro Pro Glu Trp Val Arg Tyr His Arg Ty - #r His Gly Arg Ser Ala          210              - #   215              - #   220                          - - Thr Val Trp Ser Leu Gly Val Leu Leu Tyr As - #p Met Val Tyr Gly Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Pro Phe Glu Gln Asp Glu Glu Ile Val Ar - #g Val Arg Leu Cys        Phe                                                                                             245  - #               250  - #               255             - - Arg Arg Arg Ile Ser Thr Glu Cys Gln Gln Le - #u Ile Lys Trp Cys Leu                  260      - #           265      - #           270                  - - Ser Leu Arg Pro Ser Asp Arg Pro Thr Leu Gl - #u Gln Ile Phe Asp His              275          - #       280          - #       285                      - - Pro Trp Met Cys Lys Cys Asp Leu Val Lys Se - #r Glu Asp Cys Asp Leu          290              - #   295              - #   300                          - - Arg Leu Arg Thr Ile Asp Asn Asp Ser Ser Se - #r Thr Ser Ser Ser Asn      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Glu Ser Leu                                                               - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 313                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Mus musculus                                                   - - <400> SEQUENCE: 7                                                         - - Met Leu Leu Ser Lys Ile Asn Ser Leu Ala Hi - #s Leu Arg Ala Arg        Pro                                                                               1               5 - #                 10 - #                 15             - - Cys Asn Asp Leu His Ala Thr Lys Leu Ala Pr - #o Gly Lys Glu Lys Glu                   20     - #             25     - #             30                  - - Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Le - #u Leu Gly Ser Gly Gly               35         - #         40         - #         45                      - - Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Al - #a Asp Asn Leu Pro Val           50             - #     55             - #     60                          - - Ala Ile Lys His Val Glu Lys Asp Arg Ile Se - #r Asp Trp Gly Glu Leu       65                 - # 70                 - # 75                 - # 80       - - Pro Asn Gly Thr Arg Val Pro Met Glu Val Va - #l Leu Leu Lys Lys Val                       85 - #                 90 - #                 95              - - Ser Ser Asp Phe Ser Gly Val Ile Arg Leu Le - #u Asp Trp Phe Glu Arg                  100      - #           105      - #           110                  - - Pro Asp Ser Phe Val Leu Ile Leu Glu Arg Pr - #o Glu Pro Val Gln Asp              115          - #       120          - #       125                      - - Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Le - #u Gln Glu Asp Leu Ala          130              - #   135              - #   140                          - - Arg Gly Phe Phe Trp Gln Val Leu Glu Ala Va - #l Arg His Cys His Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Gly Val Leu His Arg Asp Ile Lys Asp Gl - #u Asn Ile Leu Ile        Asp                                                                                             165  - #               170  - #               175             - - Leu Ser Arg Gly Glu Ile Lys Leu Ile Asp Ph - #e Gly Ser Gly Ala Leu                  180      - #           185      - #           190                  - - Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gl - #y Thr Arg Val Tyr Ser              195          - #       200          - #       205                      - - Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr Hi - #s Gly Arg Ser Ala Ala          210              - #   215              - #   220                          - - Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Me - #t Val Cys Gly Asp Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro Phe Glu His Asp Glu Glu Ile Ile Lys Gl - #y Gln Val Phe Phe        Arg                                                                                             245  - #               250  - #               255             - - Gln Thr Val Ser Ser Glu Cys Gln His Leu Il - #e Lys Trp Cys Leu Ser                  260      - #           265      - #           270                  - - Leu Arg Pro Ser Asp Arg Pro Ser Phe Glu Gl - #u Ile Arg Asn His Pro              275          - #       280          - #       285                      - - Trp Met Gln Gly Asp Leu Leu Pro Gln Ala Al - #a Ser Glu Ile His Leu          290              - #   295              - #   300                          - - His Ser Leu Ser Pro Gly Ser Ser Lys                                      305                 3 - #10                                                    - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 313                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Rattus norvegicus                                              - - <400> SEQUENCE: 8                                                         - - Met Leu Leu Ser Lys Ile Asn Ser Leu Ala Hi - #s Leu Arg Ala Ala Pro        1               5 - #                 10 - #                 15              - - Cys Asn Asp Leu His Ala Asn Lys Leu Ala Pr - #o Gly Lys Glu Lys Glu                   20     - #             25     - #             30                  - - Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Le - #u Leu Gly Ser Gly Gly               35         - #         40         - #         45                      - - Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Al - #a Asp Asn Leu Pro Val           50             - #     55             - #     60                          - - Ala Ile Lys His Val Glu Lys Asp Arg Ile Se - #r Asp Trp Gly Glu Leu       65                 - # 70                 - # 75                 - # 80       - - Pro Asn Gly Thr Arg Val Pro Met Glu Val Va - #l Leu Leu Lys Lys Val                       85 - #                 90 - #                 95              - - Ser Ser Gly Phe Ser Gly Val Ile Arg Leu Le - #u Asp Trp Phe Glu Arg                  100      - #           105      - #           110                  - - Pro Asp Ser Phe Val Leu Ile Leu Glu Arg Pr - #o Glu Pro Val Gln Asp              115          - #       120          - #       125                      - - Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Le - #u Gln Glu Glu Leu Ala          130              - #   135              - #   140                          - - Arg Ser Phe Phe Trp Gln Val Leu Glu Ala Va - #l Arg His Cys His Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Gly Val Leu His Arg Asp Ile Lys Asp Gl - #u Asn Ile Leu Ile        Asp                                                                                             165  - #               170  - #               175             - - Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Ph - #e Gly Ser Gly Ala Leu                  180      - #           185      - #           190                  - - Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gl - #y Thr Arg Val Tyr Ser              195          - #       200          - #       205                      - - Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr Hi - #s Gly Arg Ser Ala Ala          210              - #   215              - #   220                          - - Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Me - #t Val Cys Gly Asp Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro Phe Glu His Asp Glu Glu Ile Val Lys Gl - #y Gln Val Tyr Phe        Arg                                                                                             245  - #               250  - #               255             - - Gln Arg Val Ser Ser Glu Cys Gln His Leu Il - #e Arg Trp Cys Leu Ser                  260      - #           265      - #           270                  - - Leu Arg Pro Ser Asp Arg Pro Ser Phe Glu Gl - #u Ile Gln Asn His Pro              275          - #       280          - #       285                      - - Trp Met Gln Asp Val Leu Leu Pro Gln Ala Th - #r Ala Glu Ile His Leu          290              - #   295              - #   300                          - - His Ser Leu Ser Pro Ser Pro Ser Lys                                      305                 3 - #10                                                    - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 313                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 9                                                         - - Met Leu Leu Ser Lys Ile Asn Ser Leu Ala Hi - #s Leu Arg Ala Ala Pro        1               5 - #                 10 - #                 15              - - Cys Asn Asp Leu His Ala Thr Lys Leu Ala Pr - #o Gly Lys Glu Lys Glu                   20     - #             25     - #             30                  - - Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Le - #u Leu Gly Ser Gly Gly               35         - #         40         - #         45                      - - Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Se - #r Asp Asn Leu Pro Val           50             - #     55             - #     60                          - - Ala Ile Lys His Val Glu Lys Asp Arg Ile Se - #r Asp Trp Gly Glu Leu       65                 - # 70                 - # 75                 - # 80       - - Pro Asn Gly Thr Arg Val Pro Met Glu Val Va - #l Leu Leu Lys Lys Val                       85 - #                 90 - #                 95              - - Ser Ser Gly Phe Ser Gly Val Ile Arg Leu Le - #u Asp Trp Phe Glu Arg                  100      - #           105      - #           110                  - - Pro Asp Ser Phe Val Leu Ile Leu Glu Arg Pr - #o Glu Pro Val Gln Asp              115          - #       120          - #       125                      - - Leu Phe Asp Phe Ile Thr Glu Arg Gly Ala Le - #u Gln Glu Glu Leu Ala          130              - #   135              - #   140                          - - Arg Ser Phe Phe Trp Gln Val Leu Glu Ala Va - #l Arg His Cys His Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Gly Val Leu His Arg Asp Ile Lys Asp Gl - #u Asn Ile Leu Ile        Asp                                                                                             165  - #               170  - #               175             - - Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Ph - #e Gly Ser Gly Ala Leu                  180      - #           185      - #           190                  - - Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gl - #y Thr Arg Val Tyr Ser              195          - #       200          - #       205                      - - Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr Hi - #s Gly Arg Ser Ala Ala          210              - #   215              - #   220                          - - Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp Me - #t Val Cys Gly Asp Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Pro Phe Glu His Asp Glu Glu Ile Ile Arg Gl - #y Gln Val Phe Phe        Arg                                                                                             245  - #               250  - #               255             - - Gln Arg Val Ser Ser Glu Cys Gln His Leu Il - #e Arg Trp Cys Leu Ala                  260      - #           265      - #           270                  - - Leu Arg Pro Ser Asp Arg Pro Thr Phe Glu Gl - #u Ile Gln Asn His Pro              275          - #       280          - #       285                      - - Trp Met Gln Asp Val Leu Leu Pro Gln Glu Th - #r Ala Glu Ile His Leu          290              - #   295              - #   300                          - - His Ser Leu Ser Pro Gly Pro Ser Lys                                      305                 3 - #10                                                    - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:antisense            oligonucleotide complementary to the - #region                                surrounding the translational start - #site of HKID-1                         mRNA                                                                     - - <400> SEQUENCE: 10                                                        - - agagcagcat cgcgggcgac ggc           - #                  - #                    23                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:antisense            oligonucleotide complementary to the - #region                                surrounding the translational start - #site of HKID-1                         mRNA                                                                     - - <400> SEQUENCE: 11                                                        - - agcagcatcg cgggcgac             - #                  - #                      - #  18                                                                 __________________________________________________________________________

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. An isolated nucleic acid molecule having anucleotide sequence selected from the group consisting of:(a) Thenucleotide sequence shown in SEQ ID NO: 1; (b) A nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO:2; and (c) Anucleotide sequence complementary to either of the nucleotide sequencesin (a) or (b).
 2. The nucleic acid molecule of claim 1 furthercomprising vector nucleic acid sequences.
 3. The nucleic acid moleculeof claim 1 further comprising nucleic acid sequences encoding aheterologous polypeptide.
 4. A host cell which contains the nucleic acidmolecule of claim
 1. 5. The host cell of claim 4 which is a mammalianhost cell.
 6. A non-human mammalian host cell containing the nucleicacid molecule of claim
 2. 7. A process for producing an isolatedpolynucleotide comprising culturing a host cell transformed with avector comprising the nucleic acid of claim 1 and isolating saidpolynucleotide therefrom.
 8. A polynucleotide prepared by the process ofclaim 7.